Supplementary Figure S5 from Resistance to Durvalumab and Durvalumab plus Tremelimumab Is Associated with Functional <i>STK11</i> Mutations in Patients with Non–Small Cell Lung Cancer and Is Reversed by STAT3 Knockdown
Authors: Pore Nabendu , Wu Song , Standifer Nathan , Jure-Kunkel Maria , de los Reyes Melissa , Shrestha Yashaswi , Halpin Rebecca , Rothstein Raymond , Mulgrew Kathy , Blackmore Stephen , Martin Philip , Meekin John , Griffin Matthew , Bisha Ina , Proia Theresa A. , Miragaia Ricardo J. , Herbst Ronald , Gupta Ashok , Abdullah Shaad E. , Raja Rajiv , Frigault Melanie M. , Barrett J. Carl , Dennis Phillip A. , Ascierto Maria Libera , Oberst Michael D. 
Published: April 2023

<p>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.</p>