Identification of virus resistant tumor cell subpopulations in three-dimensional uveal melanoma cultures

Abstract

To better understand melanoma resistance to herpes simplex virus type 1 (HSV-1)-mediated oncolysis, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of OCM1 and C918 uveal melanoma cells were infected with an HSV-1 strain that expresses the green fluorescent protein (GFP) marker during replication. Although 2D cultures were completely destroyed within a few days of HSV-1 inoculation, viable GFP-negative tumor cells remained detectable in 3D cultures for several weeks. Tumor cells with increased resistance to HSV-1 included cells that formed vasculogenic mimicry patterns and multicellular spheroids and cells that invaded Matrigel individually. Mechanisms of tumor resistance against HSV-1 in the 3D environment included impaired virus spread in the ECM and ECM-mediated inhibition of viral replication after viral entry into tumor cells. Observations also suggested that HSV-1 established quiescent infection in some tumor cells present in multicellular spheroids and that this could revert to productive viral infection when the tumor growth pattern changed. These findings indicate that 3D tumor cell cultures can be used to identify distinct tumor cell populations with increased resistance to HSV-1 and to explore mechanisms of ECM-mediated tumor resistance to oncolytic virotherapy.

Increased Resistance of Vasculogenic Mimicry-Forming Uveal Melanoma Cells against Cytotoxic Agents in Three-Dimensional Cultures

“…Recent studies suggest that malignant melanoma initiating cells (MMIC) are specifically associated with VM and it has been proposed that one mechanism by which MMIC promote tumor growth is by the induction of VM formation by MMIC [17]. Recent studies in our laboratory indicate that VM-forming tumor cells in three-dimensional (3D) uveal melanoma cultures have increased resistance against cytotoxic agents and oncolytic herpes simplex virus-mediated destruction [18,19]. These observations raise the possibility that the increased therapy resistance of VM-forming tumor cells is due to a cancer stem cell phenotype.…”

“…To explore this possibility, the current study was designed to determine the expression of a known cancer stem cell marker, CD271 in traditional two-dimensional (2D) and 3D cultures of C918 uveal melanoma cells by fluorescent immunocytochemistry. C918 uveal melanoma cells have been reported to form VM in 3D cultures [18]. As controls, we also studied CD271 expression in another uveal melanoma cell line, OCM1 that does not form VM in culture [18].…”

New Methods in Tissue Engineering: Improved Models for Viral Infection

“…Latent HSV DNA can be reactivated upon UV exposure, providing an in vitro reactivation model to complement animal studies. HSV has also been studied in the context of oncolytic virus therapy in TE tumor models: Viral infection and spread depend upon ECM degradation in brain tumors (81) and melanoma models (81a). Dynamic culture systems (82) can support microorganism growth as well, with a commensal bacterium modulating epithelial barrier integrity in a TE microfluidic device (83).…”

Oncolytic HSV-1 Virotherapy: Clinical Experience and Opportunities for Progress

“…Changes in cell signaling by the tumor ECM also have been reported to induce resistance to HSV-1. In one study, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of uveal melanoma cells were infected with a GFP expressing HSV-1 [88]. Despite efficient oncolysis observed in the monolayer cultures, the same cells grown in three dimensional cultures appeared to resist oncolytic HSV-1 infection.…”