Extended Data Fig. 9: Efficacy of targeted therapy directed against Cxcr1/2 and treatment effects on immune microenvironment.

A. Kaplan-Meier plot depicting overall survival differences between patients with MDSC-high vs. MDSC-low signatures based on clustering of human TCGA PDAC samples (n = 178 patients) shown in Extended Data Fig. 2b. B. Representative images (left) of established iKRAS tumors treated with control and anti-Gr1 neutralizing antibody for 4 weeks with indicated staining. Scale bars: 100 µm. The bar graph (right) shows quantification of each cell type as analyzed by IHC. n = 6 biological replicates. Two-sided Student’s t-test. C. Tumor volume after 4 weeks of treatment with control or anti-Gr1 neutralizing antibody in mice bearing established (tumor volume ~250mm³ prior to treatment initiation) orthotopic iKRAS tumors (n = 10 mice/group). Two-sided Student’s t-test. D. Expression of Cxcr2 on granulocytic MDSCs in untreated iKRAS tumors, assessed by flow cytometry and analyzed by FlowJo (n = 3 tumors). E. Representative images of human PDAC tumors with indicated staining. Scale bars: 100 µm. Red arrow indicates positively stained cells in the same area of a core specimen. F. UMAP demonstrating cell types in single-cell RNA sequencing of human PDAC samples from Steele et al.¹⁰ with the expression of CXCR1 and CXCR2 on granulocytes/neutrophils and expression the of CSF1R, CCR2 and TREM2 on monocytes/macrophages. G. Migration of MDSCs toward conditioned medium from iKRAS tumor cells treated with control or SX-682 (n = 3 biological replicates). Student’s t-test. H. Tumor volume after 4 weeks of treatment with control or SX-682 in mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) (n = 10 mice/group). Two-sided Student’s t-test. I. Stratification of infiltrating CD4⁺ and CD8⁺ T cells as naive (CD44^lowCD62L^high), central memory (CD44^highCD62L^high) and effector memory (CD44^highCD62L^low), in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3 + anti-41BB + SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n = 3 biological replicates). Two-sided Student’s t-test. J. Quantification of total tumor associated macrophages (TAM) and dendritic cells (DC) in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3 + anti-41BB + SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n = 3 biological replicates). Two-sided Student’s t-test. K. Expression of Cxcr2 on myeloid cells in established iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or combination (anti-LAG3 + anti-41BB + SX-682) for 4 weeks assessed by flow cytometry and analyzed by FlowJo (n = 3 biological replicates). L. Representative images (left) of control, SX-682 or combination (anti-LAG3 + anti-41BB + SX-682) treated iKRAS tumors with indicated staining. Scale bars: 100 µm. The bar graphs (right) show quantification of each cell type as analyzed by IHC. n = 6 biological replicates. Two-sided Student’s t-test. M. Quantification of change in the proportion of cells in cluster M_c2 as a proportion of total monocyte/macrophage cells in scRNA-seq analysis of iKRAS tumors following treatment with control, SX-682 or combination (anti-LAG3 + anti-41BB + SX-682) for 4 weeks (n = 3 mice/group). (*p < 0.05 mixed effect model) N. Tumor volume of mice bearing established orthotopic iKRAS tumors (tumor volume ~250mm³ prior to treatment initiation) treated with control, SX-682 or SX-682 with CD8 T cell depleting antibody (n = 10 mice/group). Two-sided Student’s t-test. Data in D,G,I,J,M are presented as mean ± s.e.m.

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