Flow cytometry analysis of purity of isolated human tumor cells after mouse cell depletion.  The original cell fraction (bulk tumor) and the negative cell fraction from the depletion (isolated human tumor cells) were labeled with a pan-mouse antibody cocktail and an antibody against human CD326 (EpCAM) and analyzed by flow cytometry. Over 99% of the contaminating mouse cells were eliminated in less than 20 minutes.

Cultures of isolated tumor cells were nearly pure after seven days. Upon magnetic separation, the original bulk fraction (left) and tumor cell fraction after mouse cell depletion (right) were cultured for three to seven days, fixed, and stained. Cells were stained for the human-specific epithelial tumor marker CD326 (EpCAM) and vimentin (human tumor cells were negative for vimentin) to unambiguously identify fibroblasts. Even after seven days, the cultures of isolated tumor cells were nearly pure.

Positive influence of non-tumor cell depletion on the quality of next-generation sequencing data. DNA from bulk tumor or isolated tumor cells of three different xenograft models derived from human kidney, lung, and bladder cancer were used to produce exome-captured sequencing libraries applying the Nextera® Rapid Capture Exome Kit (Illumina®). For sequencing on a MiSeq® instrument (Illumina), the MiSeq Reagent Kit v3 (150 cycles, Illumina) was utilized to generate 75-bp paired-end reads. As the capture oligonucleotides used for targeted enrichment of protein-coding sequences were designed based on the human genome, an initial pre-enrichment of DNA fragments of human origin from the mixture of mouse and human cells was expected. To assess the number of capture oligonucleotides that might cross-hybridize with mouse genomic DNA, BLAST searches of each single Nextera probe against mouse genome were conducted and the resulting alignment parameters were used to determine possible cross-hybridization. Depending on the selection thresholds (alignment length, no. of mismatches, no. of gaps), a cross-reactivity of 5–10% of capture probes with mouse transcripts was predicted (data not shown). (A) A significant increase (p < 0.05) in cluster density (not shown) as well as an average increase in read counts of 33% was observed for the samples depleted of mouse cells, indicating improved sample quality. A corresponding strong reduction of debris and dead cells upon mouse cell depletion was also apparent in flow cytometry analysis (data not shown). (B) After adapter clipping (trimmomatic v0.32), reads of all samples were mapped against human and mouse genomes (bwa v0.7.12) and putative origin was determined based on the respective alignment parameters (LINUX shell, command-line Perl). (C) Detailed read assignment for bulk tumor and isolated human tumor cells derived from the bladder cancer xenograft. An average of 12% of reads derived from bulk tumor samples was attributed to mouse cells. This amount could be reduced to 0.3% by prior depletion of mouse cells. On average, 15% of the mouse-derived reads mapped erroneously to the human genome (1.9% of total reads) in the bulk tumor samples, demonstrating a strong positive influence of mouse cell depletion (0.04% of total reads erroneously mapped to human genome) on downstream analyses.