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Published on: July 30, 2018
In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic
R E Adamson1, A A Frazier, H Evans
1YCR Cancer Research Unit, Department of Biology, University of York , Heslington, York YO10 5DD, United Kingdom. rea3@york.ac.uk
Abstract:
Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.
Insights
Oncolytic adenovirus Ad[I/PPT-E1A] shows high specificity for prostate cancer cells. Ex vivo testing in primary human cells confirmed limited cytotoxicity and no replication in non-target cells, ensuring safety.
Area of Science:
- Oncolytic virotherapy
- Cancer gene therapy
- Adenovirus research
Background:
- Oncolytic adenoviruses offer targeted cancer cell killing.
- Ensuring specificity is crucial to prevent off-target effects and clinical complications.
Purpose of the Study:
- To assess the specificity of Ad[I/PPT-E1A] oncolytic adenovirus.
- Evaluate viral cytotoxicity and replication in primary human non-prostate cells.
- Validate a preclinical ex vivo testing model for oncolytic adenovirus safety.
Main Methods:
- Cytotoxicity assessed using MTS assays in bronchial epithelial, urothelial, vascular endothelial cells, and hepatocytes.
- Viral replication quantified via hexon immunostaining.
- Transmission electron microscopy and GFP reporter studies examined viral localization and entry.
Main Results:
- Ad[I/PPT-E1A] demonstrated no significant cytotoxicity in most non-prostate cells, except hepatocytes.
- Hepatocyte toxicity was linked to viral sequestration, not nuclear replication.
- Active viral replication was exclusively observed in prostate cells, confirming Ad[I/PPT-E1A] specificity.
Conclusions:
- Ad[I/PPT-E1A] exhibits high specificity for prostate cancer cells.
- The ex vivo testing model using primary human cells is effective for assessing oncolytic adenovirus safety.
- This approach supports the development of safer conditionally replicating oncolytic adenoviruses.
