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Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells.

Amagai Y, Fujiyuki T, Yoneda M, Shoji K, Furukawa Y, Sato H, Kai C - Sci Rep (2016)

Bottom Line: Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments.Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis.Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies.

No MeSH data available.


Related in: MedlinePlus

Unique nectin-4 expression in SW48 cells.(a) Changes in cell viability in SW48 cells after rMV-EGFP-SLAMblind inoculation at different MOIs. A WST assay was conducted on the indicated dpi. Each datum represents the mean ± SD of three independent experiments. (b) Immunocytochemical analysis of SW48 cells. Cells were stained with anti-nectin-4 pAb, followed by Alexa-Fluor-488-conjugated anti-goat IgG Ab. The cells were then fixed, stained with Hoechst 33342, and observed under a fluorescence microscope. Isotype IgG was used as the negative control. Shown are representative data of three independent experiments. Original magnification, 60× objective lens. Bar, 30 μm. (c) The graph shows the proportions of Alexa-Fluor-488-positive cells as the means ± SD of five randomly selected microscopic fields. **,††p < 0.01 compared with the isotype-IgG-stained control and DLD1 cells on Welch’s t test, respectively. (d) WST assay of sorted SW48 cells. A WST assay was conducted using either nectin-4-positive or nectin-4-negative cells after cell sorting. (e) Representative fluorescence microscopic data of three independent experiments. Cells were inoculated with rMV-EGFP-SLAMblind at MOI 2 for 72 h. Original magnification, 20× objective lens. Bar, 100 μm. (f) The graph shows the proportions of EGFP-positive cells of five randomly selected microscopic fields. The values are the means ± SD of three independent experiments. N.S., not significant on Welch’s t test. (g) Flow-cytometric analysis of intracellular nectin-4 in SW48 cells. Cell-surface and intracellular nectin-4 expression was detected with mouse anti-nectin-4 mAb (clone N4.61) and goat anti-nectin-4 pAb, respectively. Each histogram indicates the intracellular nectin-4 level in either the nectin-4-positive or nectin-4-negative population. The grey histogram indicates the IgG control for each fraction. Shown are representative data of three independent experiments. (h) Flow-cytometric analysis of cell-surface nectin-4 expression in a short-term culture of SW48 cells. Nectin-4-negative SW48 cells were sorted and cultured in FBS-containing medium for 2 h, and the gain of surface nectin-4 expression was examined. The negative control cells were stained with nectin-4 Ab without culture. The data represent the means ± SD of the nectin-4-positive cells in three independent experiments. *p < 0.05 on Welch’s t test.
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f5: Unique nectin-4 expression in SW48 cells.(a) Changes in cell viability in SW48 cells after rMV-EGFP-SLAMblind inoculation at different MOIs. A WST assay was conducted on the indicated dpi. Each datum represents the mean ± SD of three independent experiments. (b) Immunocytochemical analysis of SW48 cells. Cells were stained with anti-nectin-4 pAb, followed by Alexa-Fluor-488-conjugated anti-goat IgG Ab. The cells were then fixed, stained with Hoechst 33342, and observed under a fluorescence microscope. Isotype IgG was used as the negative control. Shown are representative data of three independent experiments. Original magnification, 60× objective lens. Bar, 30 μm. (c) The graph shows the proportions of Alexa-Fluor-488-positive cells as the means ± SD of five randomly selected microscopic fields. **,††p < 0.01 compared with the isotype-IgG-stained control and DLD1 cells on Welch’s t test, respectively. (d) WST assay of sorted SW48 cells. A WST assay was conducted using either nectin-4-positive or nectin-4-negative cells after cell sorting. (e) Representative fluorescence microscopic data of three independent experiments. Cells were inoculated with rMV-EGFP-SLAMblind at MOI 2 for 72 h. Original magnification, 20× objective lens. Bar, 100 μm. (f) The graph shows the proportions of EGFP-positive cells of five randomly selected microscopic fields. The values are the means ± SD of three independent experiments. N.S., not significant on Welch’s t test. (g) Flow-cytometric analysis of intracellular nectin-4 in SW48 cells. Cell-surface and intracellular nectin-4 expression was detected with mouse anti-nectin-4 mAb (clone N4.61) and goat anti-nectin-4 pAb, respectively. Each histogram indicates the intracellular nectin-4 level in either the nectin-4-positive or nectin-4-negative population. The grey histogram indicates the IgG control for each fraction. Shown are representative data of three independent experiments. (h) Flow-cytometric analysis of cell-surface nectin-4 expression in a short-term culture of SW48 cells. Nectin-4-negative SW48 cells were sorted and cultured in FBS-containing medium for 2 h, and the gain of surface nectin-4 expression was examined. The negative control cells were stained with nectin-4 Ab without culture. The data represent the means ± SD of the nectin-4-positive cells in three independent experiments. *p < 0.05 on Welch’s t test.

Mentions: The results described above clearly demonstrate the nectin-4-dependent infectivity and killing activity of rMV-EGFP-SLAMblind against colorectal cancer cells. Interestingly, the virus showed strong killing activity against SW48 cells (Fig. 2b), even though only approximately 15% of the population expressed nectin-4 in SW48 cells (Fig. 1a). To confirm lower MOI efficiently kills such cells with low expression of nectin-4, we inoculated the cells with three MOIs (0.1, 0.5, and 2) and found that, even at a lowest MOI, SW48 was killed efficiently (Fig. 5a). The heterogeneity of nectin-4 expression on SW48 cells was also confirmed with an immunofluorescence assay (Fig. 5b,c). To analyse whether rMV-EGFP-SLAMblind infects only the nectin-4-positive sub-population of SW48 cells, the nectin-4-positive and nectin-4-negative populations were sorted, and a WST assay was performed. The purity of either nectin-4-positive or -negative cells was >99% respectively. Surprisingly, not only the sorted nectin-4-positive single cells but also the nectin-4-negative cell population were infected with the virus, and were killed by it to the same extent (Fig. 5d,e,f). Based on the nectin-4-dependent infectivity of rMV-EGFP-SLAMblind in other cell lines, we hypothesized that SW48 cells constitutively express intracellular nectin-4 and intermittently express it on the cell surface, although most of the protein localizes in the cytoplasm. To examine this possibility, the total nectin-4 expression (both intracellular and cell-surface expression) and its cell-surface expression were determined. Live cells were stained with a mouse anti-nectin-4 monoclonal antibody (mAb) and an anti-mouse secondary Ab, fixed, permeabilized, and then stained with goat anti-nectin-4 polyclonal Ab (pAb), followed by an anti-goat secondary Ab. As expected, the total expression of nectin-4 was detected, regardless of the surface expression of nectin-4 (Fig. 5g). To directly determine whether nectin-4-negative cells become nectin-4-positive cells, the nectin-4-negative fraction of SW48 cells was sorted and cultured in foetal bovine serum (FBS)-containing medium for 2 h, and the extracellular nectin-4 expression was reanalysed. As shown in Fig. 5h, the population of cells expressing surface nectin-4 increased after culture. To examine whether this unique nectin-4 expression pattern in SW48 cells is attributable to the amino acid sequence of nectin-4, a sequence analysis was performed with mRNA obtained from SW48 cells. However, the sequence of the nectin-4-coding region, including its signal peptide, was identical to that in the GenBank database (accession number NM_030916.2) and no cell-line-specific mutation was observed (data not shown).


Oncolytic Activity of a Recombinant Measles Virus, Blind to Signaling Lymphocyte Activation Molecule, Against Colorectal Cancer Cells.

Amagai Y, Fujiyuki T, Yoneda M, Shoji K, Furukawa Y, Sato H, Kai C - Sci Rep (2016)

Unique nectin-4 expression in SW48 cells.(a) Changes in cell viability in SW48 cells after rMV-EGFP-SLAMblind inoculation at different MOIs. A WST assay was conducted on the indicated dpi. Each datum represents the mean ± SD of three independent experiments. (b) Immunocytochemical analysis of SW48 cells. Cells were stained with anti-nectin-4 pAb, followed by Alexa-Fluor-488-conjugated anti-goat IgG Ab. The cells were then fixed, stained with Hoechst 33342, and observed under a fluorescence microscope. Isotype IgG was used as the negative control. Shown are representative data of three independent experiments. Original magnification, 60× objective lens. Bar, 30 μm. (c) The graph shows the proportions of Alexa-Fluor-488-positive cells as the means ± SD of five randomly selected microscopic fields. **,††p < 0.01 compared with the isotype-IgG-stained control and DLD1 cells on Welch’s t test, respectively. (d) WST assay of sorted SW48 cells. A WST assay was conducted using either nectin-4-positive or nectin-4-negative cells after cell sorting. (e) Representative fluorescence microscopic data of three independent experiments. Cells were inoculated with rMV-EGFP-SLAMblind at MOI 2 for 72 h. Original magnification, 20× objective lens. Bar, 100 μm. (f) The graph shows the proportions of EGFP-positive cells of five randomly selected microscopic fields. The values are the means ± SD of three independent experiments. N.S., not significant on Welch’s t test. (g) Flow-cytometric analysis of intracellular nectin-4 in SW48 cells. Cell-surface and intracellular nectin-4 expression was detected with mouse anti-nectin-4 mAb (clone N4.61) and goat anti-nectin-4 pAb, respectively. Each histogram indicates the intracellular nectin-4 level in either the nectin-4-positive or nectin-4-negative population. The grey histogram indicates the IgG control for each fraction. Shown are representative data of three independent experiments. (h) Flow-cytometric analysis of cell-surface nectin-4 expression in a short-term culture of SW48 cells. Nectin-4-negative SW48 cells were sorted and cultured in FBS-containing medium for 2 h, and the gain of surface nectin-4 expression was examined. The negative control cells were stained with nectin-4 Ab without culture. The data represent the means ± SD of the nectin-4-positive cells in three independent experiments. *p < 0.05 on Welch’s t test.
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Related In: Results  -  Collection

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f5: Unique nectin-4 expression in SW48 cells.(a) Changes in cell viability in SW48 cells after rMV-EGFP-SLAMblind inoculation at different MOIs. A WST assay was conducted on the indicated dpi. Each datum represents the mean ± SD of three independent experiments. (b) Immunocytochemical analysis of SW48 cells. Cells were stained with anti-nectin-4 pAb, followed by Alexa-Fluor-488-conjugated anti-goat IgG Ab. The cells were then fixed, stained with Hoechst 33342, and observed under a fluorescence microscope. Isotype IgG was used as the negative control. Shown are representative data of three independent experiments. Original magnification, 60× objective lens. Bar, 30 μm. (c) The graph shows the proportions of Alexa-Fluor-488-positive cells as the means ± SD of five randomly selected microscopic fields. **,††p < 0.01 compared with the isotype-IgG-stained control and DLD1 cells on Welch’s t test, respectively. (d) WST assay of sorted SW48 cells. A WST assay was conducted using either nectin-4-positive or nectin-4-negative cells after cell sorting. (e) Representative fluorescence microscopic data of three independent experiments. Cells were inoculated with rMV-EGFP-SLAMblind at MOI 2 for 72 h. Original magnification, 20× objective lens. Bar, 100 μm. (f) The graph shows the proportions of EGFP-positive cells of five randomly selected microscopic fields. The values are the means ± SD of three independent experiments. N.S., not significant on Welch’s t test. (g) Flow-cytometric analysis of intracellular nectin-4 in SW48 cells. Cell-surface and intracellular nectin-4 expression was detected with mouse anti-nectin-4 mAb (clone N4.61) and goat anti-nectin-4 pAb, respectively. Each histogram indicates the intracellular nectin-4 level in either the nectin-4-positive or nectin-4-negative population. The grey histogram indicates the IgG control for each fraction. Shown are representative data of three independent experiments. (h) Flow-cytometric analysis of cell-surface nectin-4 expression in a short-term culture of SW48 cells. Nectin-4-negative SW48 cells were sorted and cultured in FBS-containing medium for 2 h, and the gain of surface nectin-4 expression was examined. The negative control cells were stained with nectin-4 Ab without culture. The data represent the means ± SD of the nectin-4-positive cells in three independent experiments. *p < 0.05 on Welch’s t test.
Mentions: The results described above clearly demonstrate the nectin-4-dependent infectivity and killing activity of rMV-EGFP-SLAMblind against colorectal cancer cells. Interestingly, the virus showed strong killing activity against SW48 cells (Fig. 2b), even though only approximately 15% of the population expressed nectin-4 in SW48 cells (Fig. 1a). To confirm lower MOI efficiently kills such cells with low expression of nectin-4, we inoculated the cells with three MOIs (0.1, 0.5, and 2) and found that, even at a lowest MOI, SW48 was killed efficiently (Fig. 5a). The heterogeneity of nectin-4 expression on SW48 cells was also confirmed with an immunofluorescence assay (Fig. 5b,c). To analyse whether rMV-EGFP-SLAMblind infects only the nectin-4-positive sub-population of SW48 cells, the nectin-4-positive and nectin-4-negative populations were sorted, and a WST assay was performed. The purity of either nectin-4-positive or -negative cells was >99% respectively. Surprisingly, not only the sorted nectin-4-positive single cells but also the nectin-4-negative cell population were infected with the virus, and were killed by it to the same extent (Fig. 5d,e,f). Based on the nectin-4-dependent infectivity of rMV-EGFP-SLAMblind in other cell lines, we hypothesized that SW48 cells constitutively express intracellular nectin-4 and intermittently express it on the cell surface, although most of the protein localizes in the cytoplasm. To examine this possibility, the total nectin-4 expression (both intracellular and cell-surface expression) and its cell-surface expression were determined. Live cells were stained with a mouse anti-nectin-4 monoclonal antibody (mAb) and an anti-mouse secondary Ab, fixed, permeabilized, and then stained with goat anti-nectin-4 polyclonal Ab (pAb), followed by an anti-goat secondary Ab. As expected, the total expression of nectin-4 was detected, regardless of the surface expression of nectin-4 (Fig. 5g). To directly determine whether nectin-4-negative cells become nectin-4-positive cells, the nectin-4-negative fraction of SW48 cells was sorted and cultured in foetal bovine serum (FBS)-containing medium for 2 h, and the extracellular nectin-4 expression was reanalysed. As shown in Fig. 5h, the population of cells expressing surface nectin-4 increased after culture. To examine whether this unique nectin-4 expression pattern in SW48 cells is attributable to the amino acid sequence of nectin-4, a sequence analysis was performed with mRNA obtained from SW48 cells. However, the sequence of the nectin-4-coding region, including its signal peptide, was identical to that in the GenBank database (accession number NM_030916.2) and no cell-line-specific mutation was observed (data not shown).

Bottom Line: Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments.Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis.Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

ABSTRACT
Oncolytic virotherapy is a distinctive antitumor therapy based on the cancer-cell-specific infectivity and killing activity of viruses, which exert a considerable antitumor effect with only a few treatments. Because colorectal cancer cells often acquire resistance to the molecular-targeted therapies and alternative treatments are called for, in this study, we evaluated the oncolytic activity against colorectal cancer cells of a recombinant measles virus (rMV-SLAMblind), which is blind to signaling lymphocytic activation molecule (SLAM) and infects target cells via nectin-4/poliovirus receptor-related 4 protein. We examined 10 cell lines including 8 cell lines that were resistant to epidermal-growth-factor-receptor (EGFR) targeted therapy. rMV-SLAMblind infected and lysed the nectin-4-positive cell lines dependently on nectin-4 expression, in spite of mutation in EGFR cascade. Tumour progression in xenograft models was also abrogated by the virus, and the infection of cancer cells in vivo by the virus was demonstrated with both flow cytometry and a histological analysis. Therefore, rMV-SLAMblind is considered a novel therapeutic agent for colorectal cancers, including those resistant to molecular-targeted therapies.

No MeSH data available.


Related in: MedlinePlus