Abstract 1672: Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune subpopulations
Authors: Brauner Janina , Agorku David , Langhammer Anne , Pohlig Lukas , Drewes Jan , Rockel Thomas Dino , Schilling Oliver , Mehlhose Stephan , Rawashdeh Wa'el Al , Bosio Andreas , Hardt Olaf 
Published: July 2017

Abstract
Immunotherapy against cancer has proven clinical efficacy and tremendous potential in multiple tumor entities. Syngeneic mouse tumor models represent the gold standard to analyze effects of immunotherapy due to their fully competent immune-repertoire. However, the amount and composition of tumor infiltrating leukocytes (TIL) is highly variable, complicating the targeted analysis of subpopulations. In particular, small subpopulations cannot be analyzed properly but may be lost in the background noise. When working with large cohort sizes, even the immune-phenotyping of TIL by flow cytometry is time consuming and data processing highly work intensive making pre-enrichment methods for sample debulking attractive.
We have established an automated workflow combining tissue dissociation with TIL specific isolation to improve and accelerate downstream analysis. Tumor dissociation was automated using the gentleMACS™Octo Dissociator and optimized for epitope conservation to overcome bias in immune-phenotyping caused by dissociation with aggressive of impure enzymes. Next, isolation of TIL was improved by developing a new CD45-specific enrichment reagent for the magnetic cell sorting (MACS) based isolation directly from dissociated tumor tissue. The whole workflow takes only about 90 min. To validate this method on starting material showing variable frequencies of TIL infiltration, we used syngeneic mouse tumors derived by injection three independent tumor cell lines. Tumors derived from injection of B16.F10 melanoma showed TIL frequencies of 2-4%, CT26.WT colon carcinoma tumors 15-21%, and 4T1 breast carcinoma tumors 32-37% among total viable cells after dissociation. Using a manual separation system, TIL were enriched to purities above 90% at yields above 70% for CT26.WT and 4T1 tumors, and purities above 80% at high yields above 95% for B16.F10 tumors. To address the need of parallelization and automation for sample processing in large cohort sizes used in in vivo studies, a fully automated version of the MultiMACS™ Cell24 Separator was developed by integrating the instrument in a liquid handling robotic platform. This new system, the MultiMACS™ X, can process 24 cell separations in parallel with minimal hands-on time. When compared to the manual system, equal purities were achieved whereas the overall yield of target cells was increased from 70% to 90% as shown in the case of CT26.WT tumors. Importantly, while the TIL enrichment significantly reduced the time and reagent costs in immune subset analysis, the composition of infiltrating immune cells was not affected, excluding the risk of introducing a bias by this method.
Taken together, we have developed an automated workflow for the isolation of TIL from mouse tumors reducing time and costs of downstream analysis while standardizing and enhancing the detection and quantification of immune cell subpopulations.
Citation Format: Janina Brauner, David Agorku, Anne Langhammer, Lukas Pohlig, Jan Drewes, Thomas Dino Rockel, Oliver Schilling, Stephan Mehlhose, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Hardt. Workflow automation and parallelization improves the isolation and analysis of tumor-infiltrating immune subpopulations [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 1672. doi:10.1158/1538-7445.AM2017-1672