Solid tumors are infiltrated by cells of non-tumor origin, including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. The amount and composition of infiltrating cells is highly variable and patient dependent, which makes analyses of primary tumor samples difficult. The contaminating cells lead to hybridization of non-tumor cell derived mRNA molecules to probes on microarrays and a significant reduction of sensitivity caused by measurement of irrelevant signals during next-generation sequencing or proteome analysis can be expected. In addition, the culture of human tumor cells is frequently hampered by fibroblasts overgrowing the target cells, which bias assays such as drug sensitivity tests. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from primary tissue. This procedure is based on the comprehensive depletion of cells of non-tumor origin by combining automated tissue dissociation and magnetic cell sorting (MACS). A negative selection strategy enables the isolation of the tumor cell population without knowledge of surface protein expression on these cells. Even tissues that initially contain low numbers of tumor cells (< 20%) reach purities of higher than 95% in less than 20 minutes. Here, we have applied this method to isolate human tumor cells from primary and metastatic ovarian carcinoma and thymoma specimens. The purified human ovarian carcinoma tumor cell fraction was further used for the isolation of CD133+ cancer stem cells. Cultivation of human tumor cells was more consistent with prior removal of contaminating stromal cells than without. Whole genome expression profiling of bulk human tumor tissue and matched purified tumor cells showed a significant reduction in stroma specific gene expression signatures while tumor specific signatures were enriched. Moreover, the comparison of expression profiles of isolated cancer stem cells to their parent population of purified tumor cells indicate that different tumor cell subpopulations could be characterized more precisely than if the unpurified fraction was used as reference. This suggests a reduced risk of misinterpretation when only purified tumor cells are evaluated or used as a control for smaller subpopulations. Whole exome sequencing of the same samples further verified the benefit of tumor cell purification from stromal contamination. The sequence read heterogeneity was dramatically reduced in the purified

samples. The reduction of non-tumor cell derived DNA, therefore, led to the detection of a higher number of tumor specific SNPs and INDELS, and consequently in a higher confidence in the results, in particular for mutations only present in a subset of tumor cells. Taken together, removal of non-tumor cells strongly improves their subsequent culture and molecular analysis of primary human tumor tissue.

Citation Format: Lena Willnow, Stefan Tomiuk, Jutta Kollet, Stefan Wild, Silvia Rüberg, Claudius Fridrich, Peter Mallmann, Frauke Alves, Philipp Ströbel, Dominik Eckardt, Andreas Bosio, Olaf Hardt. Isolation of primary human tumor cells significantly reduces bias in molecular analysis and improves culture of target cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 174.