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Experimental virus evolution in cancer cell monolayers, spheroids, and tissue explants.

ABSTRACT: Viral laboratory evolution has been used for different applications, such as modeling viral emergence, drug-resistance prediction, and therapeutic virus optimization. However, these studies have been mainly performed in cell monolayers, a highly simplified environment, raising concerns about their applicability and relevance. To address this, we compared the evolution of a model virus in monolayers, spheroids, and tissue explants. We performed this analysis in the context of cancer virotherapy by performing serial transfers of an oncolytic vesicular stomatitis virus (VSV-Δ51) in 4T1 mouse mammary tumor cells. We found that VSV-Δ51 gained fitness in each of these three culture systems, and that adaptation to the more complex environments (spheroids or explants) correlated with increased fitness in monolayers. Most evolved lines improved their ability to suppress β-interferon secretion compared to the VSV-Δ51 founder, suggesting that the selective pressure exerted by antiviral innate immunity was important in the three systems. However, system-specific patterns were also found. First, viruses evolved in monolayers remained more oncoselective that those evolved in spheroids, since the latter showed concomitant adaptation to non-tumoral mouse cells. Second, deep sequencing indicated that viral populations evolved in monolayers or explants tended to be more genetically diverse than those evolved in spheroids. Finally, we found highly variable outcomes among independent evolutionary lines propagated in explants. We conclude that experimental evolution in monolayers tends to be more reproducible than in spheroids or explants, and better preserves oncoselectivity. Our results also suggest that monolayers capture at least some relevant selective pressures present in more complex systems.

SUBMITTER: Al-Zaher A

PROVIDER: S-EPMC8134955 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAlthough coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms.MethodsUsing ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids.ResultsWe demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury.ConclusionsSARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.

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Experimental virus evolution in cancer cell monolayers, spheroids, and tissue explants.

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