Untouched Tumor Cell Isolation Allows For Subsequent Enrichment Of Rare Cancer Stem Cells And Improves NGS Analysis

By Lena Willnow, Stefan Tomiuk, Jutta Kollet, Stefan Wild, Silvia Rüberg, Claudius Fridrich, Peter Mallmann, Frauke Alves, Philipp Ströbel, Dominik Eckardt, Andreas Bosio, and Olaf Hardt

Solid tumors are vascularized and infiltrated by stromal cells. The amount and composition of these infiltrating non-tumor cells varies, for example, depending on tumor entity and stage or treatment history and may complicate sample analysis. Contaminating cells, for instance, lead to a significant reduction of sensitivity in next-generation sequencing (NGS) due to the measurement of irrelevant signals.

To overcome these limitations, we combined semi-automated tissue dissociation and automated immunomagnetic cell isolation. A negative selection strategy enables the isolation of heterogeneous tumor cells independent of specific surface markers by depletion of non-tumor cells. Here, we have applied the method to isolate human tumor cells from primary and metastatic ovarian carcinoma specimens, as well as from a thymoma sample. The purified tumor cell fraction of ovarian carcinoma was further used for the positive isolation of CD133⁺ cancer stem cells. Bulk tumor and isolated tumor cells were subjected to whole exome sequencing (WES) to assess the impact of non-tumor cell depletion on the sensitivity of downstream NGS analyses.