Abstract 570: Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy

Abstract

Abstract Untangling the complexity of programmed death-ligand 1 (PD-L1) expression within a heterogeneous tumor microenvironment is an urgent challenge in PD-1/PD-L1 immune checkpoint blockade therapy. Here, we address this challenge with a method, termed transparent tumor tomography (T3), facilitating three-dimensional (3D) visualization and spatial analysis of distributions of multiple biomarkers regarding to cancer cells, vasculature, and immune cells in context in the tumor microenvironment. With T3 analysis of transgenic mouse mammary tumors immunostained against Her2, CD45, Ki-67, CD31, and PD-L1, we reveal that PD-L1 expression within the tumor microenvironment is highly adaptable for efficiently preventing immune cell infiltration into the tumor. Stronger correlation of Her2 and PD-L1 expression in the tumor periphery where has a high CD45+ immune infiltrate density is determined by tumor-wide analysis. Also, tomographic analysis shows blood vessels expressing PD-L1 in the tumor core, where PD-L1 expression is lower. Furthermore, high-resolution T3 image localizes PD-L1 expression to a region between the endothelium and the surrounding smooth muscle cells in blood vessels. We investigate spatial pharmacokinetics of anti-PD-L1 antibody in the whole mouse mammary tumor in the context of hypoxia, CD31+ blood vessels, and target PD-L1+ cells. The 3D anti-PD-L1 antibody distribution is fit to a two-compartment pharmacokinetic model, yielding estimated distribution half-life of 4.7 min and terminal half-life of 2.5 days. We also evaluate anti-tumor immune responses after PD-L1 blockade therapy using T3. We observe broad distribution of tumor infiltrating CD3+CD8+ cytotoxic T cells in 3D tumor section following combination therapy of radiation and anti-PD-L1 antibody compared to PBS, anti-PD-L1 antibody alone, or radiation alone treatment group. Moreover, we apply T3 for immunoanalysis of whole core needle biopsy. We spatially map PD-L1 expression and granzymeB-producing CD3+CD8+ cytotoxic T cells in pre-treatment and in-treatment core needle biopsies at cellular resolution and in three dimensions. Meanwhile, T3 analysis is nondestructive, allowing secondary analysis by IHC and/or IF. We anticipate that T3 can be applied broadly to facilitate preclinical studies of immunotherapy and also find use in spatial, multiparameter analysis of patient biopsies, particularly to improve predictive testing and analysis of immune responses to tumor immunotherapy. Citation Format: Steve Seung-Young Lee, Vytautas P. Bindokas, Stephen J. Kron. Transparent tumor tomography (T3): 3D spatial immunoanalysis for PD-L1 immune checkpoint blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 570. doi:10.1158/1538-7445.AM2017-570