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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.


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In vivo DTR transduction and selection of CRC patient-derived xenografts.(a) In vivo selection of a PDX grown in a NOD-SCID mouse and directly injected (white arrow) with the DTR-GFP lentiviral vector, imaged by IVIS. After about five weeks of selection, a GFP-positive area emerges in the tumor mass. The xenograft area was shaved to reduce fur-derived background. (b) Flow cytometry analysis for GFP and human HLA-APC signals of unselected (left) or DT-selected PDXs (right), explanted at the end of the selection period. (c) Fluorescence micrograph displaying GFP expression in unselected (left) and DTR-injected, DT-selected PDX subsequently re-implanted and grown in the absence of DT (right). (d) IVIS imaging of an additional CRC PDX model in vivo injected with DTR-GFP lentiviral particles in a single or multiple site (upper and lower panels, respectively) and selected with DT for six weeks. In this model GFP-positive regions became already detectable after three weeks of selection.
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f4: In vivo DTR transduction and selection of CRC patient-derived xenografts.(a) In vivo selection of a PDX grown in a NOD-SCID mouse and directly injected (white arrow) with the DTR-GFP lentiviral vector, imaged by IVIS. After about five weeks of selection, a GFP-positive area emerges in the tumor mass. The xenograft area was shaved to reduce fur-derived background. (b) Flow cytometry analysis for GFP and human HLA-APC signals of unselected (left) or DT-selected PDXs (right), explanted at the end of the selection period. (c) Fluorescence micrograph displaying GFP expression in unselected (left) and DTR-injected, DT-selected PDX subsequently re-implanted and grown in the absence of DT (right). (d) IVIS imaging of an additional CRC PDX model in vivo injected with DTR-GFP lentiviral particles in a single or multiple site (upper and lower panels, respectively) and selected with DT for six weeks. In this model GFP-positive regions became already detectable after three weeks of selection.

Mentions: As a proof of concept of the efficiency of the DTR vector system in an additional preclinical platform, we reproduced the same experimental procedure employing human CRC PDXs instead of cell line xenografts. A preliminary test confirmed that also these PDX tumors were sensitive to DT, independently of their genetic makeup (Supplementary Fig. 7). One CRC PDX model was transduced in vivo by a single intratumoral injection of GFP-DTR lentiviral particles. Also in this case transduction efficiency was estimated as above to be around 1%. The DT selection process was then monitored by in vivo tracking of the GFP signal in the transduced tumors. To reduce the background due to the white fur, the skin surrounding the xenograft was shaved. After about five weeks of selection, the GFP signal became detectable in the tumor mass, and kept increasing in the following weeks (Fig. 4a). As confirmed by flow cytometry and microscopic analysis, almost all human cancer cells were positive for GFP (Fig. 4b,c). Also in this case lentiviral transduction of PDX tumors with GFP-DTR did not alter tumor morphology and proliferative index (Ki67 expression; Supplementary Fig. 8) or the expression of differentiation and functional markers commonly employed for CRC classification (CDK20, CDX2 and b-catenin; Supplementary Fig. 9. In addition, we found that the fraction of GFP positive cells remains very high (92%) after multiple passages of propagation of the transduced PDX in the absence of DT selective pressure (Supplementary Fig. 10).


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 DTR transduction and selection of CRC patient-derived xenografts.(a) In vivo selection of a PDX grown in a NOD-SCID mouse and directly injected (white arrow) with the DTR-GFP lentiviral vector, imaged by IVIS. After about five weeks of selection, a GFP-positive area emerges in the tumor mass. The xenograft area was shaved to reduce fur-derived background. (b) Flow cytometry analysis for GFP and human HLA-APC signals of unselected (left) or DT-selected PDXs (right), explanted at the end of the selection period. (c) Fluorescence micrograph displaying GFP expression in unselected (left) and DTR-injected, DT-selected PDX subsequently re-implanted and grown in the absence of DT (right). (d) IVIS imaging of an additional CRC PDX model in vivo injected with DTR-GFP lentiviral particles in a single or multiple site (upper and lower panels, respectively) and selected with DT for six weeks. In this model GFP-positive regions became already detectable after three weeks of selection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: In vivo DTR transduction and selection of CRC patient-derived xenografts.(a) In vivo selection of a PDX grown in a NOD-SCID mouse and directly injected (white arrow) with the DTR-GFP lentiviral vector, imaged by IVIS. After about five weeks of selection, a GFP-positive area emerges in the tumor mass. The xenograft area was shaved to reduce fur-derived background. (b) Flow cytometry analysis for GFP and human HLA-APC signals of unselected (left) or DT-selected PDXs (right), explanted at the end of the selection period. (c) Fluorescence micrograph displaying GFP expression in unselected (left) and DTR-injected, DT-selected PDX subsequently re-implanted and grown in the absence of DT (right). (d) IVIS imaging of an additional CRC PDX model in vivo injected with DTR-GFP lentiviral particles in a single or multiple site (upper and lower panels, respectively) and selected with DT for six weeks. In this model GFP-positive regions became already detectable after three weeks of selection.
Mentions: As a proof of concept of the efficiency of the DTR vector system in an additional preclinical platform, we reproduced the same experimental procedure employing human CRC PDXs instead of cell line xenografts. A preliminary test confirmed that also these PDX tumors were sensitive to DT, independently of their genetic makeup (Supplementary Fig. 7). One CRC PDX model was transduced in vivo by a single intratumoral injection of GFP-DTR lentiviral particles. Also in this case transduction efficiency was estimated as above to be around 1%. The DT selection process was then monitored by in vivo tracking of the GFP signal in the transduced tumors. To reduce the background due to the white fur, the skin surrounding the xenograft was shaved. After about five weeks of selection, the GFP signal became detectable in the tumor mass, and kept increasing in the following weeks (Fig. 4a). As confirmed by flow cytometry and microscopic analysis, almost all human cancer cells were positive for GFP (Fig. 4b,c). Also in this case lentiviral transduction of PDX tumors with GFP-DTR did not alter tumor morphology and proliferative index (Ki67 expression; Supplementary Fig. 8) or the expression of differentiation and functional markers commonly employed for CRC classification (CDK20, CDX2 and b-catenin; Supplementary Fig. 9. In addition, we found that the fraction of GFP positive cells remains very high (92%) after multiple passages of propagation of the transduced PDX in the absence of DT selective pressure (Supplementary Fig. 10).

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