Fig. 5: The Spatial-PHLEX analysis pipeline.

a Spatial-PHLEX runs multiple spatial analyses with simple input and minimal configuration. b Its density-based clustering workflow applies the DBSCAN and alpha shape algorithms to cell position data to find dense domains of a given cell type. Outputs include (i) intracluster densities of cell types, (ii) spatial cluster composition, (iii) distances of all cells to the boundary of the nearest cluster, with a negative distance assigned to cells within the spatial cluster and a positive distance to those outside, (iv) masks for clusters, and (v) overlap metrics for spatial clusters of different cell types. c Conceptual overview of Spatial-PHLEX barrier scoring and the concept of barrier fibroblasts between CD8 T cells and tumour. bottom, Illustrative barrier quantification from a shortest paths analysis. d Intracluster densities of cell types in CD8 T-cell spatial clusters in the TRACERx 100 cohort (Pan-Immune panel; n = 3878 clusters from n = 130 tumour cores, n = 2 benign tumour-adjacent cores). e Log₁₀-transformed image-level median distance-to-nearest-epithelial-cell-cluster measurements for CD8 T cells in tumour versus normal tissue (n = 139 tumour, n = 46 normal cores). Two-tailed Wilcoxon p = 0.0045. f Quantification of immune cell cluster and tumour cell cluster co-localisation through Dice scoring (n = 136 tumour cores). Two-tailed Wilcoxon p values: CD4 T cells p = 0.006, B cells p = 1e-4. g Example low and high ɑSMA⁺ fibroblast barrier score cases. Scale bar = 150 µm. h Paired violin plots of barrier scores for CD8 T cells to non-tumour and tumour epithelial cells within the same tumour cores (n = 67 cores). Paired two-tailed Wilcoxon p value shown. i Spearman correlation of barrier score vs CD8 T-cell density in the lung tumour tissue compartment (n = 121 cores). All boxplots show median and lower and upper quartile values, and whiskers extend up to 1.5*IQR above and below quartiles. h, i The all-paths adjacent barrier fraction score and tumour cores from LUAD and LUSC histologies are used. P values are represented by: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. No adjustments for multiple testing. Source data are provided as a Source Data file.