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. 2017 Nov 10;14(1):219.
doi: 10.1186/s12985-017-0888-0.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced

Takao Morinaga  1 , Thảo Thi Thanh Nguyễn  1   2 , Boya Zhong  1   2 , Michiko Hanazono  1 , Masato Shingyoji  3 , Ikuo Sekine  4 , Yuji Tada  5 , Koichiro Tatsumi  5 , Hideaki Shimada  6 , Kenzo Hiroshima  7 , Masatoshi Tagawa  8   9
Affiliations

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced

Takao Morinaga et al. Virol J. .

Abstract

Background: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity.

Methods: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses.

Results: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level.

Conclusions: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Keywords: Cleaved caspase-3; E1A; Hexon; Image cytometry; Replication-competent adenovirus.

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Conflict of interest statement

Ethics approval and consent to participate

This article does not contain any studies with human beings or animals.

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Not applicable. This article does not contain any studies with human beings or animals.

Competing interests

The authors declare that there is no conflict of interests in this research. We obtained a grant from Nichias Corporation. It is not a pharmaceutical company but a company making industrial products for building, automobiles and pipes (see http://www.nichias.co.jp/). The grant is as a kind of their mécénat activities, corporate social contributions, which is aimed to assist for medical research for intractable cancer treatments. We are thereby irrelevant to any employment, consultancy, patents or products in development or marketed products to the company. All the authors agree to publish the data included in the manuscript.

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Figures

Fig. 1
Fig. 1
AdF35/Sur-mediated cytotoxicity. a MSTO-211H cells were uninfected or infected with AdF35/Sur at a vp/cell dose as indicated, and the numbers of adherent and floating cells were counted. Average and SE bars are shown. (−): uninfected. b MSTO-211H cells were uninfected or infected with AdF35/LacZ or AdF35/Sur (104 vp/cell) and live cell numbers were counted with a trypan blue dye exclusion assay. c MSTO-211H cells were uninfected or infected with AdF35/Sur at a vp/cell as indicated for 1 day, and were stained with anti-E1A Ab and propidium iodide. d MSTO-211H cells were uninfected or infected with AdF35/Sur (104 vp/cell) as indicated and were examined for the expression levels of viral and cellular proteins with Western blot analysis. E1A and hexon showed multiple bands. Tubulin-α was used as a loading control
Fig. 2
Fig. 2
Image cytometric analysis of E1A and cleaved caspase-3. a MSTO-211H cells were uninfected or infected with AdF35/Sur (103 or 104 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. b Quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells (104 vp/cell). Averages and SE bars are shown. c Western blot analysis to detect E1A expression in MSTO-211H cells uninfected or infected with AdF35/Sur at a vp/cell dose as indicated for 24 h. (−): uninfected. d E1A intensity per cell analyzed with image cytometry. The E1A expression per cell, corresponding each dot in (a), was plotted and the dot numbers are shown in the abscissa axis. Cells showing less than 1800 (arbitrary unit) at the E1A fluorescent intensity were gated out. A red line shows E1A intensity at 2000 and the E1A intensity of positively stained cells (>2000 units) was calculated. An average unit of the intensity of the positive cells and SE are shown with statistical analysis (ANOVA)
Fig. 3
Fig. 3
Image cytometric analysis of hexon and cleaved caspase-3. a MSTO-211H cells were uninfected or infected with AdF35/Sur (103 or 104 vp/cell) for a period as indicated, stained with Ab against hexon and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. b Quantitative analysis of hexon single positive and hexon/cleaved caspase-3 double positive cells (104 vp/cell). Averages and SE bars are shown. c Hexon intensity per cell analyzed with image cytometry. The hexon expression level per cell, corresponding each dot in (a), was plotted and the cell numbers are shown in the abscissa axis. Cells showing less than 1800 (arbitrary unit) at the hexon fluorescent intensity were gated out. A red line shows hexon intensity at 2000 and the hexon intensity of positively stained cells (>2000 units) was calculated. An average unit of the intensity of the positive cells and SE are shown with statistical analysis (ANOVA)
Fig. 4
Fig. 4
Image cytometric analysis of viral and cellular proteins. a NCI-H2452 cells were uninfected or infected with AdF35/Sur (103 or 104 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. (−): uninfected. Bar graphs shows quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells in (a) (104 vp/cell). Averages and SE bars are shown. b NCI-H2452 cells were infected as shown in (a) and stained with Ab against hexon and cleaved caspase-3. Bar graph shows quantitative analysis of hexon single positive and hexon/cleaved caspase-3 double positive cells in (b) (104 vp/cell). c MSTO-211H cells were uninfected or infected with AdF35/MK (103 or 104 vp/cell) for a period as indicated, stained with Ab against E1A and cleaved caspase-3, and analyzed with Tali image-based cytometer. Bar graph shows quantitative analysis of E1A single positive and E1A/cleaved caspase-3 double positive cells in (c) (104 vp/cell)
Fig. 5
Fig. 5
Image cytometric analysis of cellular proteins. a MSTO-211H cells were uninfected or infected with AdF35/Sur (104 vp/cell), stained with Ab against cyclin E or phosphorylated histone H3 and E1A, and analyzed with Tali image-based cytometer. Representative data are shown. The red lines are tentatively drawn to distinguish stained and unstained cells. A number in image data shows a percentage of the corresponding fraction. (−): uninfected. b Quantitative analysis of E1A-, cyclin E- and phosphorylated histone H3-positive cells in (a). Averages and SE bars are shown
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