Abstract 1592: A complete workflow for the isolation of tumor-infiltrating leukocytes from human tumors and humanized mouse models
Authors: Brauner Janina , Rawashdeh Wa'el Al , Bosio Andreas , Hardt Olaf Thorsten 
Published: September 2020

Abstract
The in-depth characterization of tumor-infiltrating leukocytes (TILs) is crucial to further improve cancer immunotherapies. Since TILs generally constitute a small subpopulation of solid tumors, they can be lost in the background noise of downstream analyses such as flow cytometry or single cell sequencing. Therefore, improving pre-enrichment methods for TILs is necessary to increase the sensitivity of such studies and save resources spent on the analysis of contaminating cell populations.
We have established a reliable workflow combining automated tissue dissociation with positive selection of CD45+ TILs from human tumors as well as humanized mouse models. Human tumor samples were stored and shipped in MACS® Tissue Storage Solution, maintaining cell viability and phenotype up to 48 hours after collection. 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 or impure enzymes.
The direct isolation of TILs by MACS® Technology was optimized for the characteristics of solid tumors, such as the presence of debris and dead cells, based on a CD45-specific enrichment reagent. As an example, TIL isolation from a human colorectal carcinoma led to purities of target cells &gt;92% and yields &gt;80%. To allow for the isolation of label free TILs, we developed an isolation strategy based on the REAlease™ Technology. Again, this method is independent of the tumor entity and even tumors with low frequencies of TIL infiltration, such as an ovarian carcinoma with a frequency of 4%, could be enriched to &gt;90% purity. Upon Bead release, &gt;99% of the cells showed no residual magnetic labeling, hence TILs could be subsequently used for a second round of magnetic isolation for the isolation of multiple subpopulations. Additionally, we demonstrated that the REAlease Biotin Complex was effectively removed from the target cells as well, making them non-distinguishable from non-labeled cells before separation. Due to the removal of all labels, epitopes used for isolation are completely available for re-labeling and can be further used for any downstream application.
Taken together, we have developed a reliable workflow for the dissociation of solid tumors followed by the isolation of TILs in less than 90 minutes. We could show efficient separation performance using the standard MACS® as well as REAlease™ Technology for positive isolation of TILs. The REAlease™ Technology allows for the efficient removal of MicroBeads and the entire labeling complex, resulting in cells that are suited for a second magnetic labeling step or any kind of downstream analysis.
Citation Format: Janina Brauner, Wa'el Al Rawashdeh, Andreas Bosio, Olaf Thorsten Hardt. A complete workflow for the isolation of tumor-infiltrating leukocytes from human tumors and humanized mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1592.