p38 blockade reverses the immune suppressive microenvironment in metastatic breast cancer

The study investigated the role of p38a in regulating the outcome of the immune-tumor interaction in metastatic breast cancer. Single-cell transcriptomic analysis of the immune-TME showed that pharmacological p38 inhibition (p38i) or tumor-specific inactivation of p38a by CRISPR/Cas9 (p38KO) resulted in a less exhausted and more activated CD8+ T cell phenotype. Moreover, the myeloid cells in the TME exhibited reduced expression of immune-suppressive factors and chemotactic receptors. Overall, this study highlights a previously unrecognized p38a-driven pathway that promotes an immune suppressive TME and that therapeutic blockade of p38a has important implications for improving antitumor immunity and patient outcomes. Overall design: 4T1 mouse mammary adenocarcinoma cells were modified to express luciferase reporter. Female BALB/c mice were implanted with 4T1-luc p38a wildtype cells (p38WT) or 4T1-luc cells with p38a genetically inactivated by CRISPR-Cas9 (p38a-KO-H9). Mice with palpable tumors in the p38WT cohort (day 4) were divided in two groups and treated with vehicle control (DPBS) or p38 inhibitor (LY2228820; 30mg/kg, daily gavage) for 10 days. At day 14 post implantation, tumors from 3 mice per group of comparable sizes were pooled together and single-cell suspensions were prepared by cold protease digestion methods. The cells were stained for CD45 (pan-immune cell marker) and 7AAD (live/dead). About 100,000 Live CD45+ cells (negative for 7AAD) were sorted using BDFACSAria II. The cells were resuspended in PBS + 0.04% FBS solution at a concentration of 1000 cells per sample. Immediately following preparation, cells were provided to the Roswell Park Genomics Shared Resource (GSR) for further processing and single cell sequencing (10x Genomics).