Abstract B021: Imaging immunotherapy in the tumor microenvironment in 3D
Authors: Kron Stephen J. , Lee Steve Seung-Young , Scholten David , Bindokas Vytautas Seung-Young , Brinkman Jacqueline 
Published: February 2019

Abstract
The range of experimental and approved cancer immunotherapies is growing exponentially, creating a pressing need for assays to match patients to agents and evaluate efficacy. Immunohistochemistry (IHC) remains the gold standard for companion, predictive and on-treatment testing to evaluate immunotherapy. By detecting only one or two antigens in each two-dimensional (2D) tissue section, IHC leaves room for diagnostic and therapeutic uncertainty. IHC is satisfactory for analysis of PD-L1 immunoreactivity but fares particularly poorly as a tool to examine tumor-infiltrating lymphocytes (TILs), a complex biomarker predicting therapeutic responses to immunotherapies. IHC is also poorly suited to following recruitment of tumor-specific, activated cytotoxic T-cells, which mediate tumor regression. The ideal test would not only permit detection of checkpoint markers such as PD-L1 but also identify and enumerate TILs and report on their distribution and activity. Recently, chemical treatment of tissue to enhance transparency, combined with new methods for three dimensional fluorescent imaging, has been established as a key tool for establishing connectivity in the central nervous system. Multiple methods to render brain tissue transparent and label multiple targets with fluorescently labeled antibody probes have been reported. Similar optical clearing, staining, and imaging are beginning to be applied to cancer, but practical methods for multiplexed 3D immunolocalization in tumors have remained lacking. Toward answering the challenge of 3D imaging cytometry of the tumor immune microenvironment, we recently developed Transparent Tissue Tomography (T3), a fast and simple protocol based on lightly fixing tissue, staining with cocktails of fluorescent antibodies and then clarifying with fructose before confocal imaging. T3 is particularly well-suited to examining immune infiltrates, suggesting potential as a diagnostic tool. We have validated T3 imaging cytometry not only in mouse tumor models but also human cancer biopsies. To evaluate feasibility as a diagnostic tool, surgical biopsies from head and neck cancer patients were sampled with a core needle biopsy device, immunostained for EGFR (cancer cells), CD3 (T-cells), CD8 (cytotoxic T-cells), CD31 (tumor blood vessels) and PD-L1, rendered transparent with 80% D-fructose, mounted and then scanned by confocal microscopy to obtain 3D images. It was straightforward to distinguish samples based on differential expression of PD-L1 and evaluate whether the expression was on tumor cells and/or stroma. Further, the levels of CTL infiltration into the tumor and/or stroma was readily determined. We could also observe a unique advantage of T3 in allowing evaluation of blood vessel density, morphology and penetration into the tumor cells, which further distinguished "hot" from "cold" tumors. Using mouse models, we have examined other potential applications for T3, including mapping the distribution of both innate and adaptive immune cell infiltrates, defining the perfused and hypoxic volumes in tumors, evaluating the dynamic antitumor immune response using markers of CTL activitation in response to therapy, and as a tool for pharmacokinetics and pharmacodynamics of checkpoint blockade antibody drugs. In particular, we have applied T3 to examine the effects of radiation on the tumor cells, immune infiltrate and microvasculature and the resulting impacts on checkpoint antibody delivery and distribution. Thereby, we have better defined the conditions to enhance synergy of radiation and checkpoint blockade. Given the simple methods and rapid sample-to-answer time, we propose T3 as a practical new tool to predict and track response to checkpoint blockade from small biopsy samples.
Citation Format: Stephen J. Kron, Steve Seung-Young Lee, David Scholten, Vytautas Seung-Young Bindokas, Jacqueline Brinkman. Imaging immunotherapy in the tumor microenvironment in 3D [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B021.