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The organotypic multicellular spheroid is a relevant three-dimensional model to study adenovirus replication and
Jacques Grill1, Martine L M Lamfers, Victor W van Beusechem
1Division of Gene Therapy, Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands.
Abstract:
The use of adenoviruses for gene transfer and as oncolytic agents is currently receiving widespread attention. As specific constraints to adenovirus distribution and spread cannot be studied in cell cultures, there is a need for an in vitro three-dimensional (3D) model mimicking the in vivo biology of tumors. We studied the interactions between tumor and adenoviruses using multicellular spheroids grown from primary brain tumor material. Using beta-galactosidase and luciferase reporter genes expressed by replication-defective adenoviruses, we showed that infection was restricted to the first layer of cells. Using a replication-competent adenovirus expressing the luciferase gene, we showed that transgene expression in the spheroid was considerably enhanced and that viral spreading deep into the 3D structure took place. In addition, a tetrazolium salt-based metabolic assay could be used to compare the oncolytic activity of different concentrations of replication-competent adenoviruses. We can conclude that organotypic spheroids offer a versatile in vitro system for studying distribution, spread, and oncolysis by adenoviruses in a clinically relevant model.
Insights
Organotypic spheroids provide a 3D model to study adenovirus gene transfer and oncolytic activity. This in vitro system accurately mimics tumor biology, showing restricted infection with defective viruses but deep spread with replication-competent ones.
Area of Science:
- Oncology
- Virology
- Biotechnology
Background:
- Adenoviruses are explored for gene therapy and cancer treatment (oncolytic agents).
- Current in vitro models like cell cultures lack the complexity to study adenovirus distribution in tumors.
- A need exists for a 3D in vitro model that mimics in vivo tumor environments for adenovirus research.
Purpose of the Study:
- To investigate adenovirus interactions with brain tumors using a 3D organotypic spheroid model.
- To assess the distribution and spread of replication-defective and replication-competent adenoviruses within tumor spheroids.
- To evaluate the oncolytic efficacy of adenoviruses in a clinically relevant in vitro tumor model.
Main Methods:
- Multicellular spheroids were generated from primary brain tumor samples.
- Replication-defective adenoviruses with reporter genes (beta-galactosidase, luciferase) were used to study initial infection.
- Replication-competent adenoviruses expressing luciferase were employed to assess viral spread and transgene expression.
- A tetrazolium salt-based metabolic assay was utilized to measure adenovirus-induced oncolysis.
Main Results:
- Infection with replication-defective adenoviruses was limited to the outermost layer of tumor spheroids.
- Replication-competent adenoviruses demonstrated enhanced transgene expression and spread throughout the 3D spheroid structure.
- The metabolic assay effectively differentiated the oncolytic activity of varying concentrations of replication-competent adenoviruses.
Conclusions:
- Organotypic spheroids serve as a valuable in vitro model for studying adenovirus distribution and spread in tumors.
- This 3D model facilitates the investigation of adenovirus oncolysis in a manner relevant to clinical applications.
- The system allows for the comparison of different adenovirus concentrations and their efficacy in a complex tumor microenvironment.
