Abstract 4671: Rapid, no-wash measurement of immune checkpoint molecules expression induced by interaction with peripheral blood mononuclear cells in breast and cervical cancer cell models

Abstract

Abstract The purpose of this study was to examine the pathways involved in the induction of immune checkpoint molecules in basal breast and cervical cancer cell lines by measuring classical biomarker expression using AlphaLISA no-wash homogeneous assays. Programmed cell death-ligand 1 (PD-L1) expression increases with tumor severity in basal-like breast cancer and is enhanced in intraepithelial neoplasia and cervical cancers. Basal-breast tumors can adapt to lymphocytic infiltration by responding to heightened concentrations of interferon gamma (IFN-y) secreted by Type 1 helper T cells with upregulation of PD-L1 protein allowing the tumors to evade immune targeting and reduce the immune response. We show here that human peripheral blood mononuclear cells (PBMCs) secrete IFN-y and other cytokines in response to stimulation with CD3/CD38 Dynabeads. We then examined modulation in expression of immune checkpoint molecules (PD-L1 and others) in HCC38 (basal breast cancer-derived) and HeLa (cervical cancer) cells in response to co-culturing with activated PBMCs, treatment with conditioned media collected from activated PBMCs, and direct treatment with recombinant IFN-y. As expected, IFN-y treatment induced dose-dependent upregulation of PD-L1 protein expression in both HCC38 and HeLa cell lines, whereas the effects of conditioned media and direct co-culturing with PBMCs resulted in more complex effects which are discussed. To determine if tumor cytoarchitecture influences responsiveness to the PBMCs, biomarker expression was measured from cultures grown in both traditional adherent monolayers and in 3D spheroid cultures using Ultra-Low Attachment (ULA) microplates. Cellular health and proliferation was assessed by measuring ATP concentration with ATPlite luminescence assays and cellular imaging with viability dyes. The upregulation of PD-L1 expression was observed to be largely independent of cellular proliferation and is further shown to be dependent on signaling through the JAK/STAT pathway by probing with AlphaLISA SureFire phosphorylation assays. These data illustrate and address some of the challenges in developing a biologically relevant culture system for examining the complex mechanisms involved in tumor evasion of the innate immune response. Citation Format: Jeanine M. Hinterneder, Jen Carlstrom, Adam Carlson, Dawn Nida. Rapid, no-wash measurement of immune checkpoint molecules expression induced by interaction with peripheral blood mononuclear cells in breast and cervical cancer cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4671.