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. 2023 Jun 22;4(7):1036–1052. doi: 10.1038/s43018-023-00576-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 6 — a, Upper: Ranking of 1/hazard ratio (HR) for each Image Feature Model (IFM1 to IFM14,950) calculated by determining the positive cell frequency for one or more of 13 markers, lying within (tumor center: CT) or outside of a region 100 µm from the tumor invasive margin (IM) model (N = 40 patients). Ranking of IFM1 is indicated. IFM2 showed an HR = 0.08 (95% CI: 0.04–0.17, p = 1.91 × 10⁻⁰⁶). Lower: Heat map showing the selected markers at the tumor or margin in each combination. 14,950 total combinations were generated as the set of 4 out of 26 parameters (13 markers in 2 regions). b, Enrichment plots showing enrichment scores (ES) for positive cells per indicated marker (and their location in the tumor or at the tumor margin) based on the 16-plex Orion images, indicating whether the marker/location feature is enriched in the IFMs linked to the best hazard ratios. The green lines represent the running ES for a given marker/location as the analysis proceeds down the ranked list. The value at the peak is the final ES. The Kolmogorov-Smirnov test was used to calculate the ES and p-values (N = 40 patients). c, Regression line scatter plot showing fraction of positive cells for indicated markers from the Orion 16-plex images vs. progression-free survival (PFS, days) for 40 patients with CRC. Each dot represents measurements from a single patient. r per plot is displayed. d, Plot bootstrapping HRs from IFM1 and IFM2 (unadjusted p = 4.62 × 10⁻²⁶ and adjusted p = 6.9 × 10⁻²¹). Related to Fig. 5b. Detailed analysis is described in Methods. Pairwise two-tailed t-tests were used unless otherwise mentioned. Box and whisker represents N = 500 (random sampling), where midline = median, box limits = Q1 (25th percentile)/Q3 (75th percentile), whiskers = 1.5 inter-quartile range (IQR), and dots = outliers (>1.5IQR). e, Representative Orion IF images of cases with high IFM2 (IS = 4 in specimen C34) and low IFM2 (IS = 0 in specimen C09). Scalebars 5 mm. Higher magnification regions of interest shown in Fig. 5d. Images are from 2 representative patients/samples.

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