Abstract
Imaging-based spatial transcriptomics methods allow for the measurement of spatial determinants of cellular phenotypes but are incompatible with random barcode-based clone-tracing methods, preventing the simultaneous detection of clonal and spatial drivers. Here we report SpaceBar, which enables simultaneous clone tracing and spatial gene expression profiling with standard imaging-based spatial transcriptomics pipelines. Our approach uses a library of 96 synthetic barcode sequences that combinatorially labels each cell. Thus, SpaceBar can distinguish between clonal dynamics and environmentally driven transcriptional regulation in complex tissue contexts.
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Data availability
Processed data are available with the paper and via Github at https://github.com/grantkinsler/SpatialBarcodes.
Raw data, which consist of extremely large files, are available upon request from the authors.
Code availability
Code for analysis and figure generation are available via GitHub at https://github.com/grantkinsler/SpatialBarcodes.
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Acknowledgements
We thank C. Triandafillou and members of the Raj laboratory for helpful discussions and feedback on the work. G.K. acknowledges support from NIH Training Grant T32-CA-009140. A.R. acknowledges support from the Samuel Waxman Cancer Research Foundation, the Mark Foundation, the Melanoma Research Alliance and the Templeton Foundation (63532). R.H.B. acknowledges support from the NIH Training Grant in Computational Genomics T32HG000046 and the NIH Medical Scientist Training Program T32GM007170. S.S. is a Bakewell Foundation Innovator of the Damon Runyon Cancer Research Foundation (DRR-81-24). S.S. also acknowledges support from Wistar/Penn Skin Cancer SPORE Career Enhancement Program P50 CA261608 and the American Cancer Society (RSG-23-1152597-01-CDP).
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Authors and Affiliations
Contributions
G.K. and Y.H. designed SpaceBar, performed experiments and conducted analysis. G.K., C.F. and Y.H. performed cloning, validation and initial experiments. H.L., J.K. and M.D. performed mouse experiments, with supervision from M.H. R.V.V. designed and cloned the backbone plasmid, with supervision from S.S. R.B. assisted with panel design and spatial analysis. A.R. provided supervision and editing. G.K. and Y.H. wrote the manuscript.
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Competing interests
A.R. receives royalties related to Stellaris RNA FISH probes. A.R. serves on the scientific advisory board of Spatial Genomics. All other authors declare no competing interests.
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Nature Methods thanks Alejo Rodriguez-Fraticelli, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Madhura Mukhopadhyay, in collaboration with the Nature Methods team. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Detection of barcodes with smFISH.
Microscopy image depicting single-molecule FISH of melanoma cells transduced with a lentiviral pool containing 3 of the SpaceBar barcodes (barcodes 1, 2, and 3). DAPI is shown in gray. Probes targeting barcodes 1, 2, and 3 were coupled to Atto 674n (pseudocolored purple), Cy3 (pseudocolored orange), and Alexa Fluor 594 (pseudocolored green), respectively. Merge of all channels shown in A. Each barcode channel is shown separately in B-D.
Extended Data Fig. 2 Uniqueness of barcode combinations, clustering reduces barcode mis-detection.
A. Top 99th percentile distance between clone cells vs the number of barcodes integrated per cell. We use the value for four barcodes as the distance threshold above which the clone calling would be considered “false clones”. B. “False clone” rate reflected by the frequency of clone with above threshold size. C. Comparing the mis-assignment rate (reflected by neighbor barcode overlap) between clustered and unclustered data vs number of barcodes per cell.
Extended Data Fig. 3 Barcode abundance distribution.
A histogram that depicts the number of cells that express each of the barcodes, measured in in vitro data.
Extended Data Fig. 4 In vitro, cells of the same clone have more similar gene expression than random cells.
Depicts histograms for the mean pairwise distance in PCA space (of the top 10 PCs) of cells that belong to the same clone. The blue histogram shows the main pairwise distance for actual clones. The gray histogram depicts cells randomly assigned to clones (keeping the number of clones and cells per clone as the real distribution).
Extended Data Fig. 5 SpaceBar identifies clonal structure in several xenograft tumors.
Each panel depicts a section of a melanoma xenograft from a different mouse. Each dot depicts a barcode transcript, colored by the barcode identity. A. Tissue section 2 (a different section from the same tumor as tumor section 1 discussed throughout the main text). B-D. Depict tumor sections 3-5, respectively.
Extended Data Fig. 6 Tumor section images.
A. GFP channel from the first hybridization round of the focal xenograft section. Barcoded cells are GFP+ and autofluorescence shows the vascularized interior of the tumor. B. DAPI channel of the section.
Extended Data Fig. 7 SFRP1 clonal structure would be difficult to detect without high resolution.
A. Expression of SFRP1 in the neighborhood of Clone 31 as depicted in Fig. 2, with grids spaced 55µm apart. B. A series of heatmaps that depict the expression pattern of SFRP1 as well as the distribution of spots that label distinct clones in this area of the tumor section. Clones are depicted as the sum of all transcripts for barcodes assigned to that clone. While only clone 31 has true clonal SFRP1 gene expression from our high-resolution data, all nine depicted clones have significant correlations (p < 0.01) with SFRP1 expression when binned at 55µm resolution.
Extended Data Fig. 8 IFIT2 has high expression in nearby cells of the same clone, suggesting transiently heritable expression.
The expression of IFIT2 is shown in the tumor section. Color indicates the expression level per cell; cells with zero or one spot are depicted in gray. The first inset depicts the expression of IFIT2 in the neighborhood of Clone 72 (clone 72 cells outlined in red). The second inset depicts the expression of IFIT2 in the neighborhood of Clone 15 (clone 15 cells outlined in red). IFIT2 expression is high in groups of 3-5 cells within the same clone, suggesting its expression is transiently heritable.
Supplementary information
Supplementary Information (download PDF )
Supplementary Figures, Tables and Notes.
Supplementary Data 1 (download XLSX )
Supplementary data file which includes all of the clone and space scores calculated for the tumor sections.
Supplementary Table 1 (download XLSX )
Supplementary file which includes the barcode sequences, probe oligo sequences for smFISH, and the GenePS panel information.
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Kinsler, G., Fagan, C., Li, H. et al. SpaceBar enables single-cell-resolution clone tracing with imaging-based spatial transcriptomics. Nat Methods 23, 328–333 (2026). https://doi.org/10.1038/s41592-025-02968-w
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DOI: https://doi.org/10.1038/s41592-025-02968-w
