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. 2024 Sep 16;25(11):5113–5140. doi: 10.1038/s44319-024-00258-8

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) UMAP visualization of RNA expression clusters from single-cell sequencing analysis of tumors from two animals per treatment group (total n = 4). Tumors were harvested on day 5 after treatment started. (B) UMAP visualization of RNA expression clusters of the four individual tumors (n = 2 in each group). (C) UMAP plots showing the distribution of cells with high Notch (top) and G2M scores (bottom). Bar plots on the right quantify the depletion of clusters with the highest Notch activity (clusters 5, 8, 11) and proliferative scores (clusters 4 and 5) by combination relative to control treatment. The Y axis shows the proportion of cells in the indicated clusters relative to all cells sequenced for the respective tumor. Shown are the mean values ± interquartile ranges of the percentage of total cells in two animals in each group. (D) UMAP plots showing the distribution of cells with high qNSC-like (top) and MES-GB like scores (bottom). Bar plots on the right quantify the percentages of the cluster with high qNSC-like cell scores (cluster 11) and MES-GB scores (clusters 9, 10, and 13) by combination relative to control treatment. The Y axis shows the proportion of cells in the indicated clusters relative to all cells sequenced for the respective tumor. Shown are the mean values ± interquartile ranges of the percentage of total cells in two animals in each group. (E) Visualization of the dataset using scaled Developmental and Injury Response AUCell scores as X and Y axes, respectively. (F) Violin plot showing the AUCell scores of Development-GB and Injury-GB. n = 8160 and 17,334 cells in Vehicle and Combo groups, respectively. The box-and-whisker plot shows the interquartile range (box), the median AUCell scores (line inside the box), and the range of data within 1.5 times the interquartile range from the first and third quartiles (whiskers). P values were calculated using unpaired two-tailed Wilcoxon’s rank-sum tests.