Recombinant replication-competent viruses were demonstrated to produce cytotoxic effects preferentially on tumors and a number of clinical trials were conducted to evaluate efficacy and safety of the viral agents [
1]. Herpes simplex viruses expressing
granulocyte macrophage colony stimulating factor gene, for example, produced significant effects on metastatic melanoma and have been approved in USA and EU [
2]. Adenoviruses (Ad) are one of the agents that are relatively easy to be genetically modified and are commonly used to produce replication-restricted types targeting human tumors. Preferential Ad replication in tumors can be achieved by activation of the E1A region with a transcriptional regulatory unit of a gene which is up-regulated in human tumors [
3]. Replacing an authentic viral E1A regulatory region with such an exogenous region enable Ad to proliferate in tumors without damaging normal tissues and consequently tumor cell death was induced. We and others previously showed that a 5′ untranslated region of
survivin (Sur) [
4] or
midkine (MK) [
5] gene, which were up-regulated in the expression in a number of human tumors, activated a reporter gene in human tumors but much less in human normal cells. Replication-competent Ad powered by such a regulatory region in fact produced cytotoxicity in various type of human tumors [
4,
6]. We also developed Ad in which the fiber-knob region, mediating Ad binding to the cellular receptors [
7], was replaced with that of other subtypes. Type 5 Ad use coxsachie adenovirus receptor (CAR) as the main cellular receptor and integrin αvβ3 and αvβ5 as the ancillary receptor, whereas type 35 Ad vector use CD46 as the main receptor [
8]. Type 5 Ad bearing the Ad35-derived fiber-knob structure (AdF35) therefore infected CD46-positive cells irrespective of CAR expression [
9,
10]. An expression level of CAR molecules in human tumors is often down-regulated, whereas that of CD46 molecules was rather up-regulated in a number of human tumors [
11]. AdF35 consequently infected human tumors better than Ad5 [
12] and produced greater cytotoxicity [
13].
A mechanism of cell death induced by Ad replications can be different from that by conventional chemotherapy. Replication-competent Ad were thereby examined for a possible combinatory use with the anti-cancer agents and recently with immunotherapy [
14]. Prediction of Ad-mediated cytotoxicity will be important in a future clinical application but such a predictive biomarker remains unknown in a preclinical study. One of the reasons is a complexity of Ad-mediated cell death since viral replications and subsequent viral spreading are influenced by cellular factors which may affect infection efficacy in an interaction between tumor cells and the microenvironment, anti-viral immune responses and susceptibility of tumors to cell death [
15,
16].
Detailed analyses of viral and cellular proteins expressed are crucial for investigating viral replications and induction of cytotoxicity in target cells. Western blot analysis can show expression levels of viral and cellular proteins in a population but is not be suitable for detecting those in a small cell population. In this study, we tested a possible use of image cytometry by detecting the viral early and late proteins together with cellular proteins. Image cytometry can analyze gene expression and the levels of multiple proteins with easy. The present study demonstrated that an image cytometric technique was a handy method to monitor expression of viral and host cell proteins at a single cell level.