Fig. 7: Multimodal analysis using ST and SP in a single slide.

a Two assays were combined on the same slide and section: PhenoCycler Fusion (SP) and Xenium (ST). A segmentation mask was created using a human-in-the-loop approach and inputted into the Xenium Ranger. This mask was then transferred to the SP assay, maintaining cell IDs between the two experiments. b After segmentation, a matrix was extracted containing the pixel values of each immunofluorescent channel from the SP and the transcripts per cell from the ST. c This cell-by-feature matrix was then cell-assigned using TACIT. d. The matched number of cells assigned by the SP and ST assays was quantified to evaluate the correlation in cell assignment for each major cell type – structural (n = 25) and immune cells (n = 31). The correlation for structural cells using all transcripts and proteins was 0.37, and for immune cells, it was 0.01 (Two-sided Wilcoxon test – p-value = 2.1e-11). e. Following initial annotation, specific cell markers assigned cell types with protein and transcript designations in both proteomics and transcriptomics. The masks of cells in three high-density ROIs of immune cells showed 34% agreement with all markers used. f A smaller subset of matched protein and RNA panels was utilized to improve agreement. The Voronoi mask showed better convergence in cell type annotation, increasing cell ID matching to 81%. g, h The six cell types annotated using matched protein and RNA markers showed improved cell assignment (n = 4). The proportions were analyzed with a two-sided Wilcoxon test: *(p < 0.05), ***(p < 0.001), and NS(not significant). i After multimodal cell assignment, TACIT was also able to provide cell state markers for each cell. PD-1 and PDCD1 were used to understand the ratio of transcripts and proteins in high-density immune cell ROIs. The presence of these two markers was analyzed using SP alone, ST alone, and the two assays combined. j The proportion of positivity cell state in mRNA such as PDCD1 and MKI67 are significantly lower than PD-1 (Two-sided Wilcoxon test: p-value < 0.05, n = 4) and (k) Ki67 (Two-sided Wilcoxon test: p-value < 0.05, n = 4) in protein for B cells and CD4 + T cells across TLS. Source data are provided as a Source Data file.