(P 352) DETERMINING THE CONTRIBUTIONS OF STAT1 TO THE UPS ANTI-TUMOR MICROENVIRONMENT

Objective: Sarcoma is a heterogeneous group of cancers with over 70 subtypes and survival rates have remained relatively stagnant in recent decades. We previously found that a subset of Undifferentiated Pleomorphic Sarcoma (UPS) cases harboured tumor-infiltrating lymphocytes (TILs), expressed high levels of PD-1 ligand (PD-L1) RNA, and had better clinical outcomes than those with low PD-L1 expression. Through RNA sequencing, a list of genes differentially expressed between UPS cases with high and low PD-L1 expression was determined. Genes in the Th1 pathway were significantly overexpressed within this list. We hypothesize that tumor-related genes from the Th1 pathway may be related to an anti-tumor microenvironment. Our aims are to identify the potential roles of Th1 pathway genes, PD-L1 and STAT1, in both cell-autonomous and non-autonomous behaviours. This will be accomplished by characterizing patient-derived PD-L1 high and low cell lines followed by examining the functional differences in these cell lines both in the presence of interferon-gamma (IFN-γ) pathway activation and in the absence of STAT1. Transcriptomic changes, cytokine profile, proliferation, migration, invasion, and autologous TIL-sensitivity are being assessed.

Methods: Sarcoma specimens from patients undergoing open biopsy or surgical resection have been collected for the downstream isolation of both tumor cell lines and the respective TIL cultures. Cell lines were treated with recombinant IFN-γ and analyzed for proliferation by evaluating DNA content over time. The ability to migrate in the presence of IFN-γ was assessed by tracking cell migration across a scratch/wound over 24 hours. In addition, IFN-γ treated cells were subjected to RNA-seq. STAT1 expression in cell lines was disrupted with CRISPR-Cas9 or siRNA methodologies before IFN-γ treatment. Isogenic cell lines were assessed for transcriptomic changes in addition to changes in proliferation, and migration after IFN-γ treatment. A multiplexed Luminex assay was used to identify what cytokines are secreted from isogenic UPS cell lines at rest and in the presence of activating IFN-γ. The isogenic cell lines will be co-cultured with autologous or HLA-A,B,C blocked TIL populations to identify if a STAT1-dependent role exists in tumor-TIL interactions.

Results: Cell lines from PD-L1 high and low cases show unique rates of proliferation and migration, and this is altered by IFN-γ in some cell lines. Distinct transcriptional programs related to cytokine secretion are present in PD-L1 high cell lines after IFN-γ treatment and suggest these cells may be a source of anti-tumor cytokines. UPS cell lines have been successfully gene-edited to be deficient in STAT1 protein production, and phenotypic characterizations of the isogenic cell lines are underway.

Conclusion: Cell lines derived from PD-L1 high and low UPS cases demonstrate variable rates of proliferation and migration when untreated, however some lines show IFN-γ-dependent changes in these behaviors. Although not all characteristics are impacted by IFN-γ; the upregulation of specific transcriptional programs related to anti-tumor functions were IFN-γ-dependent. Future experiments will explore if IFN-γ pathway activation or disrupted STAT1 protein production can contribute to different cytokine profiles in cell lines, which could suggest tumor-driven microenvironment conditions. By assessing the functional consequences of IFN-γ pathway activation and STAT1-disruption, we aim to clarify how STAT1-directed cellular behaviour may be related to the improved anti-tumor microenvironment of the UPS PD-L1 high group.