Fig. 9: Combination treatment with Trastuzumab and CSF1R inhibitor induces tumor regression in ATG9A KO tumors.

A Trastuzumab-treated sgGAL4 and sgATG9A tumors were processed for single-cell RNA-Seq library preparation (6 tumors from individual sgGAL4 mice were pooled into two samples [three tumors per sample], and the same was done for 6 tumors from sgATG9A mice. 10,000 cells were analyzed per sample). B t-distributed stochastic neighbor embedding (tSNE) plot shows clustering of cells (K-Means = 2). Two major clusters of macrophages were identified. Cytotoxic macrophages, marked by elevated Nos, Cox2, Lcn2, and S100a8 expression; and regulatory macrophages, indicated by increased Cx3cr1, Cd206, Tgf-β, and Il10 expression. C Cell numbers of cytotoxic and regulatory macrophages were quantified for both sgGAL4 and sgATG9A tumors (mean ± SEM). Statistical significance was determined using two-tailed unpaired Student’s t-tests. D Violin plots show Log2 combined cytotoxic or regulatory macrophage markers expression. Violin plots display the distribution of gene expression across cells (via kernel density estimation), with an overlaid box plot showing the median and interquartile range (25th–75th percentiles); whiskers extend to the full data range (minimum and maximum values). E Cells with high csf1r expression were plotted in purple, low-csf1r-cells were shown in gray. FCsf1r high percentage in the regulatory and cytotoxic clusters from ATG9A KO tumors were quantified (mean ± SEM). Statistical significance was determined using two-tailed unpaired Student’s t-tests. scRNA-seq data was obtained from two biological replicates per condition; each replicate consisted of 10,000 cells pooled from 3 mice. While each cell provides an individual measurement, statistical comparisons between conditions should be interpreted with caution due to the limited number of pooled biological replicates (n = 2). G, H Tumor size summary of control or KO tumors under CSF1Ri alone (n = 10 tumors) or combined Trastuzumab + CSF1Ri treatment during 6 weeks (n = 30 tumors). I Summarized sgGAL4 and sgATG9A tumor growth under Trastuzumab alone (n = 25 tumors; mean ± SEM) or Trastuzumab + CSF1Ri (n = 30 tumors; mean ± SEM). Statistical significance was determined using two-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001. Some elements of this figure were created with BioRender.com and are included under a publication license in accordance with BioRender’s user agreement. Created in BioRender. DeSelm, C. (2025) https://BioRender.com/qqmbp7u.