Extended Data Fig. 1: Leiden clustering of immunity hubs.

(a–d) Paired RNA smFISH/IF and IF only stained slides were coregistered for 46 of the patients. PanCK staining is shown in cyan for RNA smFISH/IF and magenta for IF panel images. (e–g) tSNE projection as in Fig. 2a colored by (E) patient ID, (F) patient response status, and (G) immunity hub size. (h) Patient composition within each immunity hub Leiden subcluster. (i) Plot as in (H), but the y-axis depicts the number of subclusters, colored by the patient. Each column is a Leiden subcluster. (j) Frequency of immunity hub subclusters expressed as a fraction of total tumor area for responders and non-responders (NR = 33 and R = 13 patients). Two-sided Mann-Whitney test p-value is shown. (k-l) Kaplan-Meier analysis of progression-free survival (PFS) and overall survival (OS) for each subcluster. Patients were classified as either above or below a threshold of 0.1% of total tumor area for each subcluster. Shown p-values are adjusted for multiple hypothesis testing (7 tests) using the Benjamini-Hochberg method (J-L). (m) Counts of indicated phenotypes within immunity hubs, as in Fig. 2g. Phenotype counts were normalized by immunity hub area and scaled from 0-1. Each point represents the mean value across all immunity hubs of a given Leiden subcluster for each patient having that subcluster (number of patients having subcluster: #1 = 18, #2 = 33, #3 = 31, #4 = 11, #5 = 27, #6 = 25, and #7 = 30). Boxplots are defined as follows: center line = median, box minima and maxima = interquartile range (IRQ), and whiskers = 1.5*IQR. Statistical comparison was performed using an unpaired two-sided Mann-Whitney test relative to subcluster 3. Benjamini-Hochberg adjusted p-values shown: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (n) Fraction of TCF7+PD1+ CD8 T cells found within subcluster 3 in responders versus non-responders. Each dot represents one patient (NR = 33 and R = 13 patients). Two-sided Mann-Whitney test p-value is shown. (o) Counts of contact (at least touching in a cardinal direction) between TCF7+ aggregates and subcluster 3 immunity hubs versus other immunity hub subclusters (subclusters 1-2 and 4–7). Statistical comparison was performed using a two-sided Fisher’s exact test.