Abstract 5680: Quantitative cellular and molecular imaging of the intact tumor microenvironment
Authors: Martinek Jan , Brookes Hannah M. , Wu Te-chia , Sun Lili , Henrich Philipp , Kim Kyung In , George Joshy , Robson Paul , Banchereau Jacques , Palucka Karolina 
Published: August 2018

Abstract
The causes underlying the extent and character of tumor-associated immune responses in cancer are not well defined and are likely multifactorial, including cancer cell heterogeneity, host genotype, and the immune status of individual patients. Tumors are organized tissues with numerous reciprocal local and systemic connections with immune cell populations of both the myeloid and lymphoid lineages. Understanding the cellular and molecular composition of tumors could help uncover the variation in immune response to cancer in patients and to delineate interpatient patterns that could help elucidate causes of disease progression. Multiparameter flow cytometry and single-cell transcriptomics technologies have provided an unprecedented level of cell typing and annotation of cells present in tumors. However, these methods require tissue dissociation into single-cell suspensions. This is routinely linked with cell loss and artifacts due to enzymatic treatment, and cell sorting en route to transcriptomic analysis as well as cell contamination. Furthermore, tissue dissociation and single-cell analysis do not enable the determination of cellular context in which the cells find themselves at the time of analysis. Given this, we have established a comprehensive approach to cellular and molecular analysis of intact non-dissociated tissues. To this end, we have developed polychromatic (10+) immunofluorescence staining protocols for frozen tissue sections that are analyzed by confocal microscopy and whole-tissue scanning. Post-acquisition quantitation is based on adapted and improved histocytometry method based on Gerner et al. (Immunity 2012 Aug 24;37(2):364-76) with dynamic cell segmentation using Imaris software and a flow cytometry-like analysis conducted with Flowjo, allowing us to gate on different immune populations and subpopulations based on their phenotype while preserving each cell's original location within the tissue. In the next step, specific cells of interest are harvested based on their location with laser capture microdissection and profiled with RNA-seq. Transcripts derived by this computational pipeline are further analyzed using in situ hybridization with ViewRNA to confirm cell expression and establish the cellular context. We are also implementing light-sheet microscopy for 3D assessment of tissue architecture and organization by a given molecule. Thus, we are generating extensive quantitative and qualitative “maps” of cancer, stromal and immune infiltrate, allowing us to study its dynamic within the tissue.
Citation Format: Jan Martinek, Hannah M. Brookes, Te-chia Wu, Lili Sun, Philipp Henrich, Kyung In Kim, Joshy George, Paul Robson, Jacques Banchereau, Karolina Palucka. Quantitative cellular and molecular imaging of the intact tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5680.