RGB-Marking to Identify Patterns of Selection and Neutral Evolution in Human Osteosarcoma Models

Simple Summary Nowadays, we are assisting a re-discovered interest in the field of cancer heterogeneity and in defining the clonal dynamics governing tumor growth, progression, and therapy resistance. Sequencing data suggest different models of cancers development and a relationship with patients' prognosis and therapeutic response. Only a few studies have attempted to reconstruct osteosarcoma evolution, providing evidence of linear and branched pattern of clonal development. In this study, we employed a single-cell marking strategy to study the clonal dynamics of human, canine, and murine osteosarcoma models. With our collection of primary samples and cell lines, we demonstrate that different clones can evolve in parallel and generate sub-clones with similar tumorigenic potential, in a sort of extremely branched development of neutrally coexisting clones. Osteosarcoma (OS) is a highly aggressive tumor characterized by malignant cells producing pathologic bone; the disease presents a natural tendency to metastasize. Genetic studies indicate that the OS genome is extremely complex, presenting signs of macro-evolution, and linear and branched patterns of clonal development. However, those studies were based on the phylogenetic reconstruction of next-generation sequencing (NGS) data, which present important limitations. Thus, testing clonal evolution in experimental models could be useful for validating this hypothesis. In the present study, lentiviral LeGO-vectors were employed to generate colorimetric red, green, blue (RGB)-marking in murine, canine, and human OS. With this strategy, we studied tumor heterogeneity and the clonal dynamics occurring in vivo in immunodeficient NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) mice. Based on colorimetric label, tumor clonal composition was analyzed by confocal microscopy, flow cytometry, and different types of supervised and unsupervised clonal analyses. With this approach, we observed a consistent reduction in the clonal composition of RGB-marked tumors and identified evident clonal selection at the first passage in immunodeficient mice. Furthermore, we also demonstrated that OS could follow a neutral model of growth, where the disease is defined by the coexistence of different tumor sub-clones. Our study demonstrates the importance of rigorous testing of the selective forces in commonly used experimental models.

Automated summary

There is a renewed interest in cancer heterogeneity and clonal dynamics governing tumor growth, progression, and therapy resistance. Sequencing data suggest different cancer development models related to patients' prognosis and therapeutic response. Some studies have reconstructed the evolution of osteosarcoma, showing evidence of linear and branched clonal development patterns.