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Evaluating baculovirus as a vector for human prostate cancer gene therapy.

Swift SL, Rivera GC, Dussupt V, Leadley RM, Hudson LC, Ma de Ridder C, Kraaij R, Burns JE, Maitland NJ, Georgopoulos LJ - PLoS ONE (2013)

Bottom Line: Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects.BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts.BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire Cancer Research Unit, Department of Biology, University of York, Heslington, York, United Kingdom.

ABSTRACT
Gene therapy represents an attractive strategy for the non-invasive treatment of prostate cancer, where current clinical interventions show limited efficacy. Here, we evaluate the use of the insect virus, baculovirus (BV), as a novel vector for human prostate cancer gene therapy. Since prostate tumours represent a heterogeneous environment, a therapeutic approach that achieves long-term regression must be capable of targeting multiple transformed cell populations. Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects. We employed a number of prostate cancer models to analyse the potential for BV to achieve these goals. In vitro, both traditional prostate cell lines as well as primary epithelial or stromal cells derived from patient prostate biopsies, in two- or three-dimensional cultures, were used. We also evaluated BV in vivo in murine prostate cancer xenograft models. BV was capable of preferentially transducing invasive malignant prostate cancer cell lines compared to early stage cancers and non-malignant samples, a restriction that was not a function of nuclear import. Of more clinical relevance, primary patient-derived prostate cancer cells were also efficiently transduced by BV, with robust rates observed in epithelial cells of basal phenotype, which expressed BV-encoded transgenes faster than epithelial cells of a more differentiated, luminal phenotype. Maximum transduction capacity was observed in stromal cells. BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts. BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954. Thus, BV is capable of transducing a large proportion of prostate cell types within a heterogeneous 3-D prostate tumour, can facilitate cell death using a pro-drug approach, and shows promise as a vector for the treatment of prostate cancer.

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BV transduction of primary prostate epithelial cells.(A) Percentage of EGFP-positive cells at 48 h post-transduction with Bv-[CMV-EGFPCAT] at MOI = 500 for increasing lengths of time in three primary malignant prostate epithelial samples (Gleason 6, 7 or 8/9), maintained in a basal state by culture in KSFMsup (▪), or cultured in differentiating conditions in DH10 (▴). Error bars depict −/+ one standard deviation. (B) Localisation of BV capsids in primary epithelial cells from a Gleason 8/9 tumour grown in a bilayer, either 1 h or 16 h after incubation with Bv-[CMV-EGFPCAT] at MOI = 250. Red fluorescence indicates BV capsids detected with anti-vp39, nuclear DAPI staining is shown in blue and BV-driven EGFP expression in green. Confocal images of the upper layer of cells (luminal-like; at an average z-distance of 14.58 µm from the ventral position) and lower layer (basal-like; at an average z-distance of 6.47 µm from the ventral position) are shown. (C) Frequency (normalised to mock = 1%) or mean fluorescence intensity of EGFP-positive primary stromal cells derived from malignant or benign biopsies 24 h following transduction with Bv-[CMV-EGFPCAT] at MOI = 500.
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pone-0065557-g002: BV transduction of primary prostate epithelial cells.(A) Percentage of EGFP-positive cells at 48 h post-transduction with Bv-[CMV-EGFPCAT] at MOI = 500 for increasing lengths of time in three primary malignant prostate epithelial samples (Gleason 6, 7 or 8/9), maintained in a basal state by culture in KSFMsup (▪), or cultured in differentiating conditions in DH10 (▴). Error bars depict −/+ one standard deviation. (B) Localisation of BV capsids in primary epithelial cells from a Gleason 8/9 tumour grown in a bilayer, either 1 h or 16 h after incubation with Bv-[CMV-EGFPCAT] at MOI = 250. Red fluorescence indicates BV capsids detected with anti-vp39, nuclear DAPI staining is shown in blue and BV-driven EGFP expression in green. Confocal images of the upper layer of cells (luminal-like; at an average z-distance of 14.58 µm from the ventral position) and lower layer (basal-like; at an average z-distance of 6.47 µm from the ventral position) are shown. (C) Frequency (normalised to mock = 1%) or mean fluorescence intensity of EGFP-positive primary stromal cells derived from malignant or benign biopsies 24 h following transduction with Bv-[CMV-EGFPCAT] at MOI = 500.

Mentions: In order to extend our evaluation of BV into a more biologically relevant situation, we employed primary patient biopsy-derived prostate epithelial cell cultures. Since the bulk of human prostate tumours comprises cells of epithelial origin, with both undifferentiated (basal) and well-differentiated (luminal) phenotypes present, we determined whether BV was capable of targeting both epithelial cell populations at different stages of differentiation. Primary prostate epithelial cells derived from three patient tumour biopsies (Gleason scores 6, 7 and 8/9) were cultured under two different conditions: in KSFMsup medium to maintain cells in a basal state, or in DH10 medium to induce differentiation, based on work by [37]. Changes in differentiation status under these distinct culture conditions were confirmed by immunofluorescent analysis of classical differentiation markers (basal markers: cytokeratins (CK) 1, 5, 10 and 14; luminal marker: CK18), based on work by [38] (Supplementary Figure 1). On a per-patient basis, cells cultured in KSFMsup or DH10 were incubated with BV-[CMV-EGFPCAT] for increasing lengths of time and analysed by flow cytometry at 48 h. Epithelial differentiation reduced transduction frequencies overall when the cells were incubated with virus for less than 10 h: for example, in cells derived from a Gleason 6 tumour, the maximum level of infection achieved in basal (KSFMsup) cells at 4 h (45.33±3.55%) was considerably higher than the peak of infection in luminal (DH10) cells (21.69±1.78%) at 8 h (Figure 2A). Over longer periods of BV incubation, the difference in transduction levels between KSFMsup- and DH10-cultured cells became less pronounced, but remained lower in differentiated (DH10) cell populations (Figure 2A).


Evaluating baculovirus as a vector for human prostate cancer gene therapy.

Swift SL, Rivera GC, Dussupt V, Leadley RM, Hudson LC, Ma de Ridder C, Kraaij R, Burns JE, Maitland NJ, Georgopoulos LJ - PLoS ONE (2013)

BV transduction of primary prostate epithelial cells.(A) Percentage of EGFP-positive cells at 48 h post-transduction with Bv-[CMV-EGFPCAT] at MOI = 500 for increasing lengths of time in three primary malignant prostate epithelial samples (Gleason 6, 7 or 8/9), maintained in a basal state by culture in KSFMsup (▪), or cultured in differentiating conditions in DH10 (▴). Error bars depict −/+ one standard deviation. (B) Localisation of BV capsids in primary epithelial cells from a Gleason 8/9 tumour grown in a bilayer, either 1 h or 16 h after incubation with Bv-[CMV-EGFPCAT] at MOI = 250. Red fluorescence indicates BV capsids detected with anti-vp39, nuclear DAPI staining is shown in blue and BV-driven EGFP expression in green. Confocal images of the upper layer of cells (luminal-like; at an average z-distance of 14.58 µm from the ventral position) and lower layer (basal-like; at an average z-distance of 6.47 µm from the ventral position) are shown. (C) Frequency (normalised to mock = 1%) or mean fluorescence intensity of EGFP-positive primary stromal cells derived from malignant or benign biopsies 24 h following transduction with Bv-[CMV-EGFPCAT] at MOI = 500.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675042&req=5

pone-0065557-g002: BV transduction of primary prostate epithelial cells.(A) Percentage of EGFP-positive cells at 48 h post-transduction with Bv-[CMV-EGFPCAT] at MOI = 500 for increasing lengths of time in three primary malignant prostate epithelial samples (Gleason 6, 7 or 8/9), maintained in a basal state by culture in KSFMsup (▪), or cultured in differentiating conditions in DH10 (▴). Error bars depict −/+ one standard deviation. (B) Localisation of BV capsids in primary epithelial cells from a Gleason 8/9 tumour grown in a bilayer, either 1 h or 16 h after incubation with Bv-[CMV-EGFPCAT] at MOI = 250. Red fluorescence indicates BV capsids detected with anti-vp39, nuclear DAPI staining is shown in blue and BV-driven EGFP expression in green. Confocal images of the upper layer of cells (luminal-like; at an average z-distance of 14.58 µm from the ventral position) and lower layer (basal-like; at an average z-distance of 6.47 µm from the ventral position) are shown. (C) Frequency (normalised to mock = 1%) or mean fluorescence intensity of EGFP-positive primary stromal cells derived from malignant or benign biopsies 24 h following transduction with Bv-[CMV-EGFPCAT] at MOI = 500.
Mentions: In order to extend our evaluation of BV into a more biologically relevant situation, we employed primary patient biopsy-derived prostate epithelial cell cultures. Since the bulk of human prostate tumours comprises cells of epithelial origin, with both undifferentiated (basal) and well-differentiated (luminal) phenotypes present, we determined whether BV was capable of targeting both epithelial cell populations at different stages of differentiation. Primary prostate epithelial cells derived from three patient tumour biopsies (Gleason scores 6, 7 and 8/9) were cultured under two different conditions: in KSFMsup medium to maintain cells in a basal state, or in DH10 medium to induce differentiation, based on work by [37]. Changes in differentiation status under these distinct culture conditions were confirmed by immunofluorescent analysis of classical differentiation markers (basal markers: cytokeratins (CK) 1, 5, 10 and 14; luminal marker: CK18), based on work by [38] (Supplementary Figure 1). On a per-patient basis, cells cultured in KSFMsup or DH10 were incubated with BV-[CMV-EGFPCAT] for increasing lengths of time and analysed by flow cytometry at 48 h. Epithelial differentiation reduced transduction frequencies overall when the cells were incubated with virus for less than 10 h: for example, in cells derived from a Gleason 6 tumour, the maximum level of infection achieved in basal (KSFMsup) cells at 4 h (45.33±3.55%) was considerably higher than the peak of infection in luminal (DH10) cells (21.69±1.78%) at 8 h (Figure 2A). Over longer periods of BV incubation, the difference in transduction levels between KSFMsup- and DH10-cultured cells became less pronounced, but remained lower in differentiated (DH10) cell populations (Figure 2A).

Bottom Line: Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects.BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts.BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954.

View Article: PubMed Central - PubMed

Affiliation: Yorkshire Cancer Research Unit, Department of Biology, University of York, Heslington, York, United Kingdom.

ABSTRACT
Gene therapy represents an attractive strategy for the non-invasive treatment of prostate cancer, where current clinical interventions show limited efficacy. Here, we evaluate the use of the insect virus, baculovirus (BV), as a novel vector for human prostate cancer gene therapy. Since prostate tumours represent a heterogeneous environment, a therapeutic approach that achieves long-term regression must be capable of targeting multiple transformed cell populations. Furthermore, discrimination in the targeting of malignant compared to non-malignant cells would have value in minimising side effects. We employed a number of prostate cancer models to analyse the potential for BV to achieve these goals. In vitro, both traditional prostate cell lines as well as primary epithelial or stromal cells derived from patient prostate biopsies, in two- or three-dimensional cultures, were used. We also evaluated BV in vivo in murine prostate cancer xenograft models. BV was capable of preferentially transducing invasive malignant prostate cancer cell lines compared to early stage cancers and non-malignant samples, a restriction that was not a function of nuclear import. Of more clinical relevance, primary patient-derived prostate cancer cells were also efficiently transduced by BV, with robust rates observed in epithelial cells of basal phenotype, which expressed BV-encoded transgenes faster than epithelial cells of a more differentiated, luminal phenotype. Maximum transduction capacity was observed in stromal cells. BV was able to penetrate through three-dimensional structures, including in vitro spheroids and in vivo orthotopic xenografts. BV vectors containing a nitroreductase transgene in a gene-directed enzyme pro-drug therapy approach were capable of efficiently killing malignant prostate targets following administration of the pro-drug, CB1954. Thus, BV is capable of transducing a large proportion of prostate cell types within a heterogeneous 3-D prostate tumour, can facilitate cell death using a pro-drug approach, and shows promise as a vector for the treatment of prostate cancer.

Show MeSH
Related in: MedlinePlus