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.

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癌细胞单层、球体和组织外植体中的实验性病毒进化

病毒实验室进化已用于不同的应用，例如模拟病毒出现、耐药性预测和治疗性病毒优化。然而，这些研究主要在细胞单层中进行，这是一种高度简化的环境，引起了人们对其适用性和相关性的担忧。为了解决这个问题，我们比较了模型病毒在单层、球体和组织外植体中的进化。我们通过在 4T1 小鼠乳腺肿瘤细胞中连续转移溶瘤性水泡性口炎病毒 (VSV-Δ51)，在癌症病毒疗法的背景下进行了这项分析。我们发现 VSV-Δ51 在这三个培养系统中的每一个都获得了适应度，并且适应更复杂的环境（球体或外植体）与单层适应度的增加相关。与 VSV-Δ51 创始人相比，大多数进化系提高了抑制 β-干扰素分泌的能力，这表明抗病毒先天免疫施加的选择性压力在三个系统中很重要。但是，也发现了特定于系统的模式。首先，在单层中进化的病毒仍然比在球体中进化的病毒更具肿瘤选择性，因为后者显示出对非肿瘤小鼠细胞的伴随适应。其次，深度测序表明，在单层或外植体中进化的病毒种群往往比在球状体中进化的病毒种群更具遗传多样性。最后，我们发现在外植体中繁殖的独立进化系之间的结果差异很大。我们得出结论，单层的实验进化往往比球体或外植体更具可重复性，并更好地保持肿瘤选择性。我们的结果还表明，单分子层至少捕获了更复杂系统中存在的一些相关选择压力。