P02.23.B MOLECULAR BARCODING OF GLIOBLASTOMA IN HUMAN ORGANOTYPIC CORTICAL SLICE CULTURE

Abstract BACKGROUND Glioblastoma are hallmarked by clonal evolution and transcriptional heterogeneity linked to genetic and environmental diversity resulting in therapy resistance and disease progression. In this study, we integrate heritable molecular barcoding and spatial transcriptomics to enable high-resolution cell lineage tracing in the spatial context of the glioblastoma TME. MATERIAL AND METHODS Patient-derived glioblastoma cells from two individual cases were isolated from anatomically and pathologically distinct regions: the primary tumor core, the tumor periphery, and the recurrent lesion. Each subline was transduced with predefined FlexTrack lentiviral plasmids encoding GFP, resulting in uniquely barcoded (9N) cell lines. Immediately before injection, the barcoded glioblastoma populations were pooled and introduced into ex vivo human cortical slices. Live-imaging was used to trace GFP-positive, barcoded cell populations. After incubation, the fresh-frozen slices were processed for spatial transcriptomics and the line-specific barcodes were mapped according to the 3′ poly(A) tails of captured transcripts. Graph neural networks based on single-cell deconvolution were employed to spatially analyze the cellular ecosystems. RESULTS We successfully deconvoluted and reconstructed the trajectories of injected glioblastoma cells linking their transcriptome phenotypes along with clonal identity. Multi-omic integration of spatial copy-number alterations and barcoding based pseudo-trajectories enabled reconstruction of the temporal transcriptional dynamics linked to the heterogeneity of tumor ecosystems. CONCLUSION This study serves as a proof-of-concept for integrating molecular barcoding and spatial transcriptomics in cortical slice culture. Our approach to molecular barcoding in complex model systems opens new avenues to investigate the contextual dynamics of clonal evolution in glioblastoma.