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A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.

Picco G, Petti C, Trusolino L, Bertotti A, Medico E - Sci Rep (2015)

Bottom Line: DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth.DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers.This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

View Article: PubMed Central - PubMed

Affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.

ABSTRACT
We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

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In vivo selection of DT transduced tumors.(a) Schema of the in vivo DTR transduction and selection experiment. (b) Growth curves of HCT116 xenografts in CD1-nude mice transduced in vivo by intratumoral injection of scramble-GFP or DTR-GFP concentrated lentiviral particles. A week after vector injection, DT was administered at increasing concentrations for three weeks, followed by 12 days of drug withdrawal and two additional weeks of treatment. One control tumor for each vector was allowed to grow in the absence of DT as a control. (c,d) Flow cytometry analysis of the fraction of GFP+ cells in unselected (c) or DT-selected (d) tumors. (e) Fluorescence micrograph displaying GFP expression in a representative DTR-injected, DT-selected HCT116 xenograft subsequently re-implanted and grown in the absence of DT.
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f3: In vivo selection of DT transduced tumors.(a) Schema of the in vivo DTR transduction and selection experiment. (b) Growth curves of HCT116 xenografts in CD1-nude mice transduced in vivo by intratumoral injection of scramble-GFP or DTR-GFP concentrated lentiviral particles. A week after vector injection, DT was administered at increasing concentrations for three weeks, followed by 12 days of drug withdrawal and two additional weeks of treatment. One control tumor for each vector was allowed to grow in the absence of DT as a control. (c,d) Flow cytometry analysis of the fraction of GFP+ cells in unselected (c) or DT-selected (d) tumors. (e) Fluorescence micrograph displaying GFP expression in a representative DTR-injected, DT-selected HCT116 xenograft subsequently re-implanted and grown in the absence of DT.

Mentions: Generation of genetically modified xenografts is easily accomplishable with most neoplastic cell lines by in vitro transduction and selection, followed by implant in mice. In the case of PDXs however, such procedure is typically not applicable or poorly efficient. We therefore sought to verify if direct intratumoral injection of the DTR vector in xenografts from human cell lines and patient-derived tumors, followed by DT treatment of mice, could lead to the development of xenografts significantly enriched in transduced cells. To this aim, HCT116 xenografts in nude mice were directly transduced by intratumoral injection of concentrated lentiviral particles of GFP-DTR or GFP-control (scramble) vector. After one week, cell suspensions were obtained from a first set of transduced tumors for flow cytometry analysis, which detected a fraction of GFP+ cells around 1% for both vector types (average of 5 measurements = 0.97% +/− 1.70%). In a parallel set of xenografts, one week after transduction, mice were treated with DT for three weeks, followed by two weeks of suspension, after which DT was maintained until tumor explant (Fig. 3a). While all tumors displayed marked shrinkage after three weeks of DT treatment, only DTR-transduced tumors resumed growth after the initial shrinkage. The regrown tumors featured a multinodular mass, suggestive of parallel growth of multiple resistant subclones from the areas of vector injection (Fig. 3b, photo insert). GFP-DTR-transduced and selected xenografts revealed a striking enrichment in human GFP+ cells with respect to transduced tumors grown in the absence of selection (99% vs 3%; Fig. 3b,c).


A diphtheria toxin resistance marker for in vitro and in vivo selection of stably transduced human cells.

Picco G, Petti C, Trusolino L, Bertotti A, Medico E - Sci Rep (2015)

In vivo selection of DT transduced tumors.(a) Schema of the in vivo DTR transduction and selection experiment. (b) Growth curves of HCT116 xenografts in CD1-nude mice transduced in vivo by intratumoral injection of scramble-GFP or DTR-GFP concentrated lentiviral particles. A week after vector injection, DT was administered at increasing concentrations for three weeks, followed by 12 days of drug withdrawal and two additional weeks of treatment. One control tumor for each vector was allowed to grow in the absence of DT as a control. (c,d) Flow cytometry analysis of the fraction of GFP+ cells in unselected (c) or DT-selected (d) tumors. (e) Fluorescence micrograph displaying GFP expression in a representative DTR-injected, DT-selected HCT116 xenograft subsequently re-implanted and grown in the absence of DT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588510&req=5

f3: In vivo selection of DT transduced tumors.(a) Schema of the in vivo DTR transduction and selection experiment. (b) Growth curves of HCT116 xenografts in CD1-nude mice transduced in vivo by intratumoral injection of scramble-GFP or DTR-GFP concentrated lentiviral particles. A week after vector injection, DT was administered at increasing concentrations for three weeks, followed by 12 days of drug withdrawal and two additional weeks of treatment. One control tumor for each vector was allowed to grow in the absence of DT as a control. (c,d) Flow cytometry analysis of the fraction of GFP+ cells in unselected (c) or DT-selected (d) tumors. (e) Fluorescence micrograph displaying GFP expression in a representative DTR-injected, DT-selected HCT116 xenograft subsequently re-implanted and grown in the absence of DT.
Mentions: Generation of genetically modified xenografts is easily accomplishable with most neoplastic cell lines by in vitro transduction and selection, followed by implant in mice. In the case of PDXs however, such procedure is typically not applicable or poorly efficient. We therefore sought to verify if direct intratumoral injection of the DTR vector in xenografts from human cell lines and patient-derived tumors, followed by DT treatment of mice, could lead to the development of xenografts significantly enriched in transduced cells. To this aim, HCT116 xenografts in nude mice were directly transduced by intratumoral injection of concentrated lentiviral particles of GFP-DTR or GFP-control (scramble) vector. After one week, cell suspensions were obtained from a first set of transduced tumors for flow cytometry analysis, which detected a fraction of GFP+ cells around 1% for both vector types (average of 5 measurements = 0.97% +/− 1.70%). In a parallel set of xenografts, one week after transduction, mice were treated with DT for three weeks, followed by two weeks of suspension, after which DT was maintained until tumor explant (Fig. 3a). While all tumors displayed marked shrinkage after three weeks of DT treatment, only DTR-transduced tumors resumed growth after the initial shrinkage. The regrown tumors featured a multinodular mass, suggestive of parallel growth of multiple resistant subclones from the areas of vector injection (Fig. 3b, photo insert). GFP-DTR-transduced and selected xenografts revealed a striking enrichment in human GFP+ cells with respect to transduced tumors grown in the absence of selection (99% vs 3%; Fig. 3b,c).

Bottom Line: DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth.DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers.This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

View Article: PubMed Central - PubMed

Affiliation: Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Torino, Italy.

ABSTRACT
We developed a selectable marker rendering human cells resistant to Diphtheria Toxin (DT). The marker (DT(R)) consists of a primary microRNA sequence engineered to downregulate the ubiquitous DPH2 gene, a key enzyme for the biosynthesis of the DT target diphthamide. DT(R) expression in human cells invariably rendered them resistant to DT in vitro, without altering basal cell growth. DT(R)-based selection efficiency and stability were comparable to those of established drug-resistance markers. As mice are insensitive to DT, DT(R)-based selection can be also applied in vivo. Direct injection of a GFP-DT(R) lentiviral vector into human cancer cell-line xenografts and patient-derived tumorgrafts implanted in mice, followed by systemic DT administration, yielded tumors entirely composed of permanently transduced cells and detectable by imaging systems. This approach enabled high-efficiency in vivo selection of xenografted human tumor tissues expressing ectopic transgenes, a hitherto unmet need for functional and morphological studies in laboratory animals.

No MeSH data available.


Related in: MedlinePlus