Abstract 2693: MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy
Authors: Longley Jemma , Foxall Russell , Thirdborough Steven , Beers Steve , Cragg Mark 
Published: March 2024

Abstract
Background: CD163 positive tumor associated macrophages (TAM), have been associated with poor response to immunochemotherapy in DLBCL. Targeting these TAMs to reverse immunosuppression is one approach for improving treatment response rates. MicroRNAs have the potential to modify hundreds of genes by interacting with multiple mRNA targets, thereby altering the global transcriptional profile of cells. Manipulation of microRNAs may prove an effective means of switching the transcriptomic profiles of TAMs to more activated, anti-tumor states resulting in the reversal of immune resistance and enhancement of treatment efficacy.
Methods: MicroRNAs differentially expressed in DLBCL and associated with poor response were identified using whole gene correlated network analysis of DLBCL datasets. Peripheral blood mononuclear cells (PBMCs) were obtained from normal blood donors and differentiated into macrophages following cell culture with MCSF. Macrophages were polarized using immunosuppressive (IL4 + IL13) or immunostimulating (LPS + IFNγ) cytokines and microRNA expression measured using RT-qPCR. To assess antibody dependent cellular phagocytosis (ADCP), macrophages were generated and polarized as above and transfected with microRNA inhibitors (Thermofisher) prior to co-incubation with target cells. Human Chronic Lymphocytic Leukemia (CLL) cells were labelled with CSFE and used as target cells with Rituximab as the ADCP-inducing monoclonal antibody (mAb) and Herceptin as an isotype control. The percentage of Fc gamma receptor IIIA (FcγRIIIA)-expressing macrophages taking up target cells was determined using flow cytometry and analyzed using Flowjo software. Diagnostic biopsies from patients were then analyzed for microRNA 142-5p and CD206 expression using miRNAScope assays and immunohistochemistry.
Results: There was significant upregulation of microRNA 142-5p in macrophages polarized with IL4 plus IL13 compared to macrophages polarized with LPS plus IFNγ and non-polarized macrophages. Inhibition of microRNA 142-5p in IL4 plus IL13 polarized macrophages led to a significant increase in FcγRIIIA, CD40 and CD47 expression resulting in enhanced phagocytosis of CLL cells opsonized with Rituximab plus anti-CD47 mAb when compared with controls. High microRNA 142-5p expression in combination with high CD206 expression predicted poor response to immunochemotherapy in 38 treatment naïve human DLBCL samples (median PFS 12 months v NR p=0.0096).
Discussion: Transfection of a microRNA 142-5p inhibitor can repolarize immunosuppressive macrophages to a more anti-tumor phenotype leading to enhanced ADCP. CD206 positive TAM infiltration in combination with high microRNA 142-5p expression predicted poor outcome following treatment with immunochemotherapy in DLBCL. It may be possible to use microRNA directed therapies targeting the tumor microenvironment to enhance treatment efficacy.
Citation Format: Jemma Longley, Russell Foxall, Steven Thirdborough, Steve Beers, Mark Cragg. MicroRNA manipulation of macrophage polarization in DLBCL to augment antibody immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2693.