FIGURE 5 from Characterizing Neutrophil Subtypes in Cancer Using scRNA Sequencing Demonstrates the Importance of IL1β/CXCR2 Axis in Generation of Metastasis-specific Neutrophils

Alistair S Mclaren,Andrew D Campbell,Cassie J Clarke,Colin Nixon,Colin W Steele,Declan Whyte,Jim Norman,John Falconer,Kathryn Gilroy,Kristina Kirschner,Leo M Carlin,Mark White,Megan L Mills,Owen J Sansom,Rachel A Ridgway,Rana Fetit,Rene Jackstadt,Renee Njunge,Tamsin R.M Lannagan,Varushka Naiker,Xabier Cortes-Lavaud

doi:10.1158/2767-9764.25315145.v1

Abstract

Loss of neutrophil-specific CXCR2 attenuates suppression of T-cell proliferation in neutrophils from the metastatic niche in mice bearing tumors. Neutrophils from the liver and blood of littermate mice either Mrp8-Cre-Cxcr2+/+ (WT) or Mrp8-Cre-Cxcr2fl/fl (CXCR2fl) mice were harvested 28 days following intrasplenic injection of villinCreER KrasG12D/+Trp53fl/flRosa26N1CD/+ (KPN) cells. These were placed in coculture with T cells from WT mice, and analyzed through flow cytometry 44 hours later. A, Flow cytometry gating of T cells and neutrophils from coculture. Cells were gated on FSC-A/SSC-A (not shown), FCS-A/FSC-H for singlets, Live/Dead, CD3, and CD4/CD8 to define CD3+, CD3+/CD4+, and CD3+/CD8+ populations for analysis. Cell trace yellow allows for tracking of T-cell proliferation. The signal for each T-cell division becomes subsequently dimmer, and allows for calculation of the number of T-cell generations. Gating obtained from CXCR2fl mouse (blue) and unstimulated T cell control (red). B, Proportion of CD3+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers (B–J, four replicates from 2 WT mice and six replicates from 3 CXCR2fl mice). C, Expansion index of CD3+ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. D, Division index of CD3+ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. E, Proportion of CD4+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. F, Expansion index of CD4+ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. G, Division index of CD4+ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. H, Proportion of CD8+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. I, Expansion index of CD8+ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. J, Division index of CD8+ T cells in coculture with WT and CXCR2fl neutrophils from tumor-bearing livers. K, Proportion of CD3+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice (K–S, three replicates from 2 WT mice and four replicates from 2 CXCR2fl mice). L, Expansion index of CD3+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. M, Division index of CD3+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. N, Proportion of CD4+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. O, Expansion index of CD4+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. P, Division index of CD4+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. Q, Proportion of CD8+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. R, Expansion index of CD8+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. S, Division index of CD8+ T cells that proliferated in coculture with WT and CXCR2fl neutrophils from blood of tumor-bearing mice. *, P < 0.05 on unpaired t test. **, P < 0.01 on unpaired t test.

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