We found that, after 40 serial passages in p53-knockout mouse embryonic fibroblasts, VSV exhibited significantly increased INCB18424 JAK inhibitor fitness and cytotoxicity in these cells, but that these changes tended to be non-adaptive in normal MEFs, therefore indicating increased selectivity for p53-deficient cells. However, full-length sequencing did not reveal simple molecular signatures underlying this phenotype. Finally, we also demonstrate p53-dependent oncolytic activity in tumor cell cultures and in vivo using mouse 4T1 breast and CT-26 colon cancer models. VSV provides a flexible platform for the design of oncolytic viruses. Nearly all of the approximately 30 different oncolytic VSVs reported in the literature have been produced by genetic engineering, such as introduction of specific mutations in the M and G proteins, generation of pseudotyped viruses expressing the surface protein of other RNA viruses, insertion of microRNAs, or insertion of genes encoding tumor suppressor, suicide or immunomodularory proteins. In contrast, very few studies have used evolutionary tools to try to increase the tumor selectivity of VSV. In one such study, an engineered pseudotyped VSV encoding a single-chain antibody against the Her2/neu receptor was found to yield low titer in target mammary cancer cells expressing ErbB2, and directed evolution was then used to improve viral fitness in these cells. In another study, VSV was serially Paclitaxel company passaged in human glioblastoma cells to select for more efficient cell attachment, faster replication, and reduced affinity for normal human fibroblasts. The virus rapidly evolved the desired properties and was later shown to be effective against other tumor cell lines. Here, we undertook a more general, experimental evolution approach by serially passaging multiple independent evolution lines in cells deficient for p53 function, a feature shared by many cancers. Based on previous work showing extensive parallel evolution in experimentally evolved VSV, we expected that some substitutions should appear repeatedly after the 40 serial passages.