PO-219 New cell genetic tracing and single-cell tools for the study of the molecular pathways involved in cancer metastasis

Introduction Traditionally, metastasis has been seen as the final and often fatal step in the progression of solid malignancies. This vision of tumour progression has been recently challenged, as genetic analyses of circulating tumour cells and functional studies in animal models, have suggested that the dissemination of tumour cells can be a very early event, even at a premalignant stage. Whether this implies that the colonisation of the distant tissue happens at such an early stage is still unclear. In accordance with an early metastatic implantation model, there is evidence in cancer of early parallel evolution of primary and metastatic tumours as well as tissue specific evolutionary branches among different metastases from the same tumour. Transcriptional analysis of cells with different metastatic potential in mouse tissues have been used to identify the genes and pathways involved in metastasis specificity. Moreover, intratumour heterogeneity has been observed in multiple cancers and has been postulated as a critical aspect for tumour metastasis and treatment resistance. In this context, the use of new molecular and sequencing strategies like cell lineage tracing systems and single-cell sequencing, to genetically modified mouse models, could provide new opportunities to unravel the molecular mechanisms behind metastatic potential. Material and methods In order to construct a new fluorescent-based lineage tracing system, we have performed a systematic evaluation of the fluorescent characteristics of different proteins and have tried several orientation and locations of loxP sites in order to provide a random and proportional repertoire of fluorescent labelling after CRE recombination. Additionally, we have set up droplet-based microfluidic technology to perform single-cell sequencing on murine tumour primary samples. Results and discussions Here we have constructed a new allele able to produce up to 15 different colour combinations that can be uniquely identified by confocal microscopy and FACS. Additionally, we have set up the infrastructure and protocol to perform single-cell RNA-Seq and targetted sequencing on tumour primary samples. Conclusion We have generated very promising new tools that could open new opportunities to study the molecular mechanisms behind metastatic potential in mouse and human tumours.