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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.

No MeSH data available.


Related in: MedlinePlus

DTR transduced cells are resistant to DT in vivo.(a,b) Tumor growth curves of xenografts from HCT116 cells transduced with either the Scramble vector (a) or the DTR vector (b) in nude mice treated with DT (1 μg/kg or 5 μg/kg) or vehicle (n = 5). (c) Schema of the in vivo selection experiment. Nude mice xenografts obtained from a mixture of DTR-transduced (GFP-positive) and parental (GFP-negative) HCT116 cells (1:20 ratio) were treated with DT (5 μg/kg) or vehicle for three weeks. It is expected that xenografts grown in the presence of DT are enriched in GFP-positive cells. (d) Tumor growth curves (n = 4) in the course of the DT selection process, followed by flow-cytometry analysis of GFP levels in representative tumors explanted from unselected (up) and selected (bottom) cohorts, as indicated.
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f2: DTR transduced cells are resistant to DT in vivo.(a,b) Tumor growth curves of xenografts from HCT116 cells transduced with either the Scramble vector (a) or the DTR vector (b) in nude mice treated with DT (1 μg/kg or 5 μg/kg) or vehicle (n = 5). (c) Schema of the in vivo selection experiment. Nude mice xenografts obtained from a mixture of DTR-transduced (GFP-positive) and parental (GFP-negative) HCT116 cells (1:20 ratio) were treated with DT (5 μg/kg) or vehicle for three weeks. It is expected that xenografts grown in the presence of DT are enriched in GFP-positive cells. (d) Tumor growth curves (n = 4) in the course of the DT selection process, followed by flow-cytometry analysis of GFP levels in representative tumors explanted from unselected (up) and selected (bottom) cohorts, as indicated.

Mentions: To assess if DTR-transduced cells maintain DT resistance also in the context of a living organism, HCT116 cells transduced with DTR or with a control vector were grown as subcutaneous xenografts in nude mice. When xenografts reached ~50 mm3, mice were treated with DT or with vehicle for three weeks. As shown in Fig. 2a, treatment of control HCT116 xenografts with 1 μg/kg DT strongly reduced their growth rate, and 5 μg/kg DT induced complete tumor regression, which persisted also after DT suspension. Conversely, DTR transduced cells resisted to both doses of DT and continued growing in the presence of DT and after its withdrawal (Fig. 2b). Notably, in the absence of DT, DTR-transduced HCT116 xenografts displayed a growth rate similar to that of control xenografts (Fig. 2a,b, “Vehicle” lines), confirming that DTR expression has no major effects on cancer cell growth. As previously reported, no adverse effects were observed in mice treated with DT1024.


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)

DTR transduced cells are resistant to DT in vivo.(a,b) Tumor growth curves of xenografts from HCT116 cells transduced with either the Scramble vector (a) or the DTR vector (b) in nude mice treated with DT (1 μg/kg or 5 μg/kg) or vehicle (n = 5). (c) Schema of the in vivo selection experiment. Nude mice xenografts obtained from a mixture of DTR-transduced (GFP-positive) and parental (GFP-negative) HCT116 cells (1:20 ratio) were treated with DT (5 μg/kg) or vehicle for three weeks. It is expected that xenografts grown in the presence of DT are enriched in GFP-positive cells. (d) Tumor growth curves (n = 4) in the course of the DT selection process, followed by flow-cytometry analysis of GFP levels in representative tumors explanted from unselected (up) and selected (bottom) cohorts, as indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: DTR transduced cells are resistant to DT in vivo.(a,b) Tumor growth curves of xenografts from HCT116 cells transduced with either the Scramble vector (a) or the DTR vector (b) in nude mice treated with DT (1 μg/kg or 5 μg/kg) or vehicle (n = 5). (c) Schema of the in vivo selection experiment. Nude mice xenografts obtained from a mixture of DTR-transduced (GFP-positive) and parental (GFP-negative) HCT116 cells (1:20 ratio) were treated with DT (5 μg/kg) or vehicle for three weeks. It is expected that xenografts grown in the presence of DT are enriched in GFP-positive cells. (d) Tumor growth curves (n = 4) in the course of the DT selection process, followed by flow-cytometry analysis of GFP levels in representative tumors explanted from unselected (up) and selected (bottom) cohorts, as indicated.
Mentions: To assess if DTR-transduced cells maintain DT resistance also in the context of a living organism, HCT116 cells transduced with DTR or with a control vector were grown as subcutaneous xenografts in nude mice. When xenografts reached ~50 mm3, mice were treated with DT or with vehicle for three weeks. As shown in Fig. 2a, treatment of control HCT116 xenografts with 1 μg/kg DT strongly reduced their growth rate, and 5 μg/kg DT induced complete tumor regression, which persisted also after DT suspension. Conversely, DTR transduced cells resisted to both doses of DT and continued growing in the presence of DT and after its withdrawal (Fig. 2b). Notably, in the absence of DT, DTR-transduced HCT116 xenografts displayed a growth rate similar to that of control xenografts (Fig. 2a,b, “Vehicle” lines), confirming that DTR expression has no major effects on cancer cell growth. As previously reported, no adverse effects were observed in mice treated with DT1024.

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