Intratumoral genetic reprogramming of murine melanoma engineers an inflamed tumor microenvironment, generates tertiary lymphoid structures, and forms CD4-CD8-APC triads

Summary: Abstract Body: This study examines local administration of reprogramming nanoparticles (NPs) on the melanoma tumor microenvironment (TME) in mice via application of a multiplex immunofluorescence (mIF) PhenoCycler panel that we previously developed for profiling murine TMEs. We administered biodegradable NPs that co-delivered 4-1BBL/IL-12 or GFP (control) plasmids intratumorally to B16F10 murine melanoma flank tumors with systemic anti-PD1 in a flank and lung metastasis model. We generated a tissue microarray from flank tumors, stained/imaged tissues, and analyzed proteomics data using QuPath and the FlowSOM R package. Initial analysis of flank tumors found that 4-1BBL/IL-12 NPs with anti-PD1 decreased melanoma cell density 2.3-fold (p=0.0001) and increased intratumoral immune cell infiltration 2.3-fold (p=0.0002), with increases in CD4 T-cell density (p=0.0001), CD8 T-cell density (p=0.0001), CD8/Treg ratio (p=0.0007), M1/M2 macrophage ratio (p=0.0001), dendritic cell (DC) density (p=0.0004), and natural killer cell density (p=0.0031). mIF images identified 7 total stromal tertiary lymphoid structures (TLSs) across all flank tumor sections treated with 4-1BBL/IL-12 NPs and anti-PD1 compared to 0 TLSs in control sections. We also found significantly increased antigen presentation markers (LMP2 and beta-2 microglobulin) on tumor cells, M1 macrophages, and dendritic cells following 4-1BBL/IL-12 NPs and anti-PD1. Additionally, 4-1BBL/IL-12 NPs and anti-PD1 induced 7.04 intratumoral CD4/CD8/proinflammatory antigen-presenting cell (M1 macrophages and DCs) triads/mm2 in flank tumors compared to 0.02 triads/mm2 in control sections (p=0.0005). These data suggest that 4-1BBL/IL-12 NPs and anti-PD1 potentiate a pro-inflammatory TME and coordinate an adaptive anti-tumor immune response through TLS generation, antigen presentation, and colocalized efforts of helper/effector T cells. Xin Ming Zhou¹, Charles Lu¹, Kathryn Luly¹, Erick Rocher¹, Sachin Surwase¹, Elizabeth Will¹, Jordan Green¹, Stephany Tzeng¹, Joel Sunshine¹ 1. The Johns Hopkins University School of Medicine, Baltimore, MD, United States. Bioinformatics, Computational Biology, and Imaging