Abstract LB-138: Complete workflows allow comprehensive tumor microenvironment analysis and culture of cell subsets of limited tumor patient samples
Authors: Richter Anne , Siemer Ramona , Criado-Moronati Elvira , Baranska Anna , Gert Philipp , Agorku David , Hardt Olaf , Vorholt Daniela , Singh Aparajita , Dose Christian , Heemskerk Bianca , Evaristo Cesar 
Published: August 2019

Abstract
Immunotherapy approaches that engage T cells to attack tumors have proven clinical efficacy and tremendous potential in multiple cancers. However, there are several mechanisms of resistance to current therapeutic strategies: clinical benefit is experienced by only a subset of patients, and often partial responses are achieved. Thus, additional research is necessary to improve immunotherapeutic strategies. In particular, it is important to analyze steady-state anti-tumor immunity and monitor the effects of therapeutic interventions on tumor-infiltrating leukocytes (TILs) and other cell populations within the tumor microenvironment. However, the phenotypic and functional analysis of tumor-infiltrating leukocytes (TILs) is technically challenging and labor intensive. The number of TILs can be very low and small subpopulations might escape analysis as they get lost in the background noise. Importantly, flow cytometry phenotyping of different cell populations requires dividing the limited tumor material into multiple samples for analysis using population-specific antibody panels, which dramatically reduces the number of cells available for analysis. Therefore, it is fundamental to use innovative tools and workflows to maximize the amount and quality of the data obtained from limited tumor patient samples. We established complete workflows combining tissue storage, dissociation, cell isolation and analysis. Tissues were stored and shipped in a solution that was shown to maintain cell viability and phenotype up to 48h after collection (Tissue Storage Solution™). Tumor dissociation was automated and optimized for epitope preservation using a tissue dissociator (gentleMACS™ Octo). We developed new enrichment reagents for magnetic cell sorting, incorporating novel technology enabling the removal of both superparamagnetic beads and antibody fragments (REAlease®). Using MACS® and REAlease technology, tumor-infiltrating T-, B-, Myeloid cells, cancer-associated fibroblasts and tumor cells were sequentially isolated. The isolated cell populations were characterized by flow cytometry, multi-parameter imaging and transcriptomics. Isolated tumor and T cells were cultured and expanded in vitro, allowing identification of tumor cell-specific T cells. Importantly, purification of tumor-infiltrating T cells with high viability was essential for high-resolution immune profiling by single cell transcriptomics. In conclusion, we have optimized workflows that allow the comprehensive analysis of the tumor microenvironement, including T-, B-, Myeloid cells, cancer associated fibroblasts and tumor cells. Sequential isolation of the different cell types maximized the number of cells of each cell type available for analysis and cultureThese workflows greatly reduce experimental time and allow the performance of more complex experimental setups. We believe the use of these innovative tools and workflows can significantly increase the quality of the data obtained in immuno-oncology and immunotherapy research.
Citation Format: Anne Richter, Ramona Siemer, Elvira Criado-Moronati, Anna Baranska, Philipp Gert, David Agorku, Olaf Hardt, Daniela Vorholt, Aparajita Singh, Christian Dose, Bianca Heemskerk, Cesar Evaristo. Complete workflows allow comprehensive tumor microenvironment analysis and culture of cell subsets of limited tumor patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-138.