The Effect of Autophagy Induction in Oncolytic Reovirus Replication in Mesenchymal Stem Cells

Background and Aims: Oncolytic reoviruses can infect and kill malignant cells while sparing their normal counterparts. Reoviral infection can induce or activate autophagy, even though metformin can induce autophagy. Identifying and regulating the cellular pathways important for reovirus replication and oncolysis can improve targeted-biological therapies for cancer. Here, the autophagic process was triggered via metformin, and we investigated the effect of autophagy activation on oncolytic reovirus replication in mesenchymal stem cells as primary cells and L929 cell lines. Materials and Methods: Adipose derived mesenchymal stem cells (AD-MSCs) and L929 cells were treated with metformin and reovirus type-3 strain Dearing (T3D). Twenty-four hours after infection, the viability of AD-MSCs and L929 cells were examined by MTT assay. Also, the effect of metformin-induced autophagy in the reovirus replication in these cells was determined by real-time polymerase-chain-reaction. Results: Our results show that treatment with metformin and reovirus reduced the viability of the cells compared to treatment with metformin or reovirus alone in both cells. Also, coadministration of metformin and reovirus significantly decreased the relative expression level of the Beclin-1 gene compared to treatment with metformin in both cells. However, the expression level of the reovirus L3 gene after treatment with metformin and reovirus in L929 cells increased significantly compared to AD-MSCs. Conclusion: Our data suggest that metformin-induced autophagy enhances reoviral replication in AD-MSCs and L929 cells. These findings represent the role of autophagy induction in facilitating reovirus replication and contribute to a better understanding of reovirus-host interactions.

Development of a reverse transcription loop mediated isothermal amplification assay for the detection of Mouse reovirus type 3 in laboratory mice

Mouse reovirus type 3 (Reo-3) infection is a viral disease that is harmful for laboratory mice. No rapid and accurate detection methods are currently available for this infection. In this study, we describe a rapid, simple, closed-tube, one step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for Reo-3 and compare our assay with indirect enzyme-linked immunosorbent assay (ELISA). Three sets of RT-LAMP primers were designed by sequence analysis of a specific conserved sequence of the Reo-3 S1 gene. Using RS2 primer set, the RT-LAMP assay required 60 min at 65 °C to amplify the S1 gene in one step by using Reo-3 RNA template and had no cross-reactivity with the other related pathogens, such as Sendai virus (SV), pneumonia virus of mice (PVM), mouse hepatitis virus (MHV), Ectromelia virus (Ect), minute virus of mice (MVM), P. pneumotropica, B. bronchiseptica, K. pneumonia and P. aeruginosa. in our LAMP reaction system. The limit of detection (LOD) of our RT-LAMP assay is 4 fg/μL. The established RT-LAMP assay enabled visual detection when fluorescence detection reagents were added, and was demonstrated to be effective and efficient. We tested 30 clinical blood samples and five artificial positive samples from SPF mice, the concordance between the two methods for blood samples was 100% compared with indirect ELISA and RT-PCR. Considering its performance, specificity, sensitivity, and repeatability, the developed RT-LAMP could be a valuable tool to supply a more effective Reo-3 detection method in laboratory animal quality monitoring.

CEACAM6 as a candidate biomarker for pelareorep sensitivity in pancreatic adenocarcinoma (PDAC).

746 Background: Pelareorep is a proprietary formulation of live, replication-competent, naturally occurring Reovirus Type 3 Dearing strain. A randomized phase II trial of pelareorep in combination with carboplatin and paclitaxel in first-line treatment of metastatic PDAC (NCT01280058) was performed. Although pelareorep did not improve the primary endpoint of progression-free survival compared to carboplatin and paclitaxel alone, impressive durable responses were seen in the pelareorep arm in some patients (pts). Further, prior studies have noted the immunomodulatory carcinoembryonic antigen-related cell adhesion molecule (CEACAM6/CD66c) as a receptor for specific viral subtypes. We thus speculated that altered CEACAM6 levels may be predictive for pelareorep sensitivity. Methods: Pre-treatment tissue biopsies were collected prior enrolment for all 73 pts on study. Evaluable pts with transcriptomic data was available for only 31 pts. RNA was purified from FFPE tissue and gene expression analysis was performed using SensationPlus FFPE Amplification and WT labelling kit and the Human Transcriptome Array 2.0. CEACAM6 protein expression was determined by immunohistochemistry. Differential gene expression and survival analysis using were performed in R/Bioconductor. Appropriate corrections for multiplicity were performed. Results: When comparing extraordinary responders in the pelareorep treated arm to those with poor outcomes, low levels of CEACAM6 mRNA expression were associated with prolonged PFS in pelareorep-treated pts (adjusted p = 0.05). This effect was not seen in non-pelareorep treated pts. The luminal, but not the cytoplasmic immunohistochemistry score, was highly correlated with mRNA expression levels of CEACAM6, p = 0.001. Modulation of CEACAM6 in vitro and in vivo are underway. Conclusions: CEACAM6 may be a candidate biomarker of sensitivity to pelareorep and, in theory, could improve viral trafficking of this compound in tumor cells. Clinical trial information: NCT01280058 . [Table: see text]

Oncolytic reovirus shows potentially anticancer effect against CT26 cell lines

Background: Reovirus type 3 Dearing (T3D) is a segmented double-stranded RNA that selectively replicates and triggers anticancer responses in the KRAS mutant cancer cells. Here, we examine the anticancer activity of ReoT3D against murine colon carcinoma cell line (CT26) with deferent in vitro experiments. Results: Our results showed that ReoT3D significantly induced oncolysis in the CT26 cells by induction of apoptosis, and cell cycle arrest in the G0/G1 and G2/M checkpoints and reduction of cell migration and colony forming ability. Conclusion: Present study confirmed that ReoT3D has strong anticancer impacts on CT26 cells which can be considered as a new approach against KRAS mutant cancer cell lines in laboratory investigation.