This research seeks to develop an imaging technique for 3D mapping of immune contexts within a tumor. Capability to determine location, density, and functional orientation of the different immune cell populations within a whole tumor would be a powerful tool to examine crosstalk between a tumor microenvironment and immune system but also to advance development of effective cancer immunotherapy strategies. Although immunofluorescence staining has long been used in cancer immunology, the conventional immunofluorescence method is restricted to the localization of targeted antigens in limited two-dimensional (2D) tumor sections or small tumor volumes. Thus, 2D immunofluorescence cannot provide integrated spatial information about the immune cell distributions or other features of tumor tissue, leaving room to misinterpret the extent or significance of responses to therapy or downstream effects. To overcome these challenges, we developed a three-dimensional (3D) immunofluorescence tomography approach to map whole tumors at single cell resolution. Thus, we optimized optical clearing of tumor macroslices, increased penetration of antibodies to permit immunofluorescence staining, applied multicolor 3D microscopy imaging, enabled tomographic reconstruction of the images, and implemented automated image analysis. This approach allows us to spatially and quantitatively determine the distribution of tumor-infiltrating T cells (CD4+, CD8+, and regulatory (CD4+CD25+Foxp3+) T cells) in whole mouse mammary (TUBO) tumors. To demonstrate utility in evaluating immune responses, we performed intratumoral injection of the STING agonist DMXAA and evaluated anti-tumor immune responses in the whole tumor, including detecting damage to the microvasculature using the CD31 endothelial marker and increase in tumor-infiltrating CD8+ T cells. Our results establish a new method, Transparent Tumor Tomography (T3), that provides integrated spatial information regarding immune contexts in whole tumors as a new tool for cancer immunotherapy research.
Citation Format: Steve Seung-Young Lee, Vytas Bindokas, Stephen J. Kron. Transparent tumor tomography (T3): Spatial 3D mapping of immune responses in a whole tumor after immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B103.
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