Supplementary Figure S5 from Resistance to Durvalumab and Durvalumab plus Tremelimumab Is Associated with Functional STK11 Mutations in Patients with Non–Small Cell Lung Cancer and Is Reversed by STAT3 Knockdown
Year: 2023
Type: supplementary-materials
Abstract: Characterization of innate and adaptive immune cell recruitment to CT26 STK11 KO tumors compared to CT26 WT. (A) Work flow for single cell RNA (scRNA) analysis of CD45+ cells isolated from CT26 WT and STK11 KO clones. Data analysis including cell quality control and filtering steps used to subset the raw scRNA-seq data into CD45+ cells and the two main immune cell populations subsequently analyzed as described in the Supplementary Appendix. (B) Number of CD45+ immune cells per sample used for downstream analysis. (C) Uniform Manifold Approximation and Projection (UMAP) of CD45+ immune cells colored by major cell populations as determined by Louvain clustering: B cell, monocyte/macrophage/DC (Mono.Macro.DC), neutrophil, T cell/NK cell (T.NK), basophil, and pDC clusters are represented as indicated. (D) UMAP plots showing the expression of key genes used in the identification of immune cell clusters. Ptprc = CD45, immune cell marker; Cd3e = epsilon subunit of CD3 complex, T cell marker; Ncr1 = NKp46, NK cell marker; Csf1r = CSF1 receptor, monocyte/macrophage marker; Csf3r = CSF3 (G-CSF) receptor, neutrophil/gMDSC marker; Siglech, Siglec H, plasmacytoid DC marker; CD19, B cell marker; Mcpt8 = mast cell protease 8, basophil/mast cell marker. (E) Percentage of each cell cluster among CD45+ immune population per sample. (F) Comparison between median percentage of immune cell clusters for CT26 WT and STK11 KO clones as indicated. *p < 0.05, one-way ANOVA. (G) Levels of intratumoral granulocytic (Ly6G+) cells as a fraction of CD45+ immune cells in WT and STK11 KO CT26 tumors as determined by flow cytometry. (H) UMAP representation showing single-cell RNA subclustering of the monocyte/macrophage/DC sub-populations as determined by Louvain clustering. (I) Top highly expressed genes between monocyte/macrophage/DC sub-clusters used for annotation as labeled in K and L. (J) Percentage of monocyte/macrophage/DC sub-populations per sample. (K) Comparison between median percentage of cells that are present in each monocyte/macrophage/DC cluster among CT26 WT and STK11 KO clones. *p < 0.05, one-way ANOVA comparing CT26 STK11 KO clones to CT26 WT. (L) Hierarchical clustering of monocyte/macrophage subpopulations (Mono.Macro) based on M1 and M2 gene signatures, as defined by Orecchioni M, et al (5). Genes were also subjected to hierarchical clustering. (M) Levels of intra-tumoral CD163+ tumor-associated macrophages determined by IHC. (N) CD45neg tumor and non-immune stromal cell RNA levels of chemokines and chemokines associated with recruitment and suppressive function of MDSCs and TAMs and of TMEM173/STING.
Authors: Nabendu Pore, Song Wu, Nathan Standifer, Maria Jure-Kunkel, Melissa de los Reyes, Yashaswi Shrestha, Rebecca Halpin, Raymond Rothstein, Kathy Mulgrew, Stephen Blackmore, Philip Martin, John Meekin, Matthew Griffin, Ina Bisha, Theresa A. Proia, Ricardo J. Miragaia, Ronald Herbst, Ashok Gupta, Shaad E. Abdullah, Rajiv Raja, Melanie M. Frigault, J. Carl Barrett, Phillip A. Dennis, Maria Libera Ascierto, Michael D. Oberst
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4362559973