Abstract 2184: Enrichment and isolation of tumor cells using a unique flow-sorting approach to decrypt intratumoral heterogeneity in matched samples of squamous cell carcinoma of the lung

Abstract

Abstract Background: Genomic intratumoral heterogeneity in cancers is a driving force for progression or recurrence. Availability of pure tumor cell material is critical for studying clonal evolution. In general, tumors are highly mixed with benign cells with a mean tumor cell purity of only 49%1 shown in lung squamous cell carcinoma (LSCC). Using a unique flow-sorting based approach enables us to overcome this limitation by highly enriching for tumor cells. Here, we aimed at precisely identifying different tumor populations in primary LSCC and matched distant metastases from 18 patients. Methods: Nuclei from snap-frozen and FFPE tumor specimens were extracted and subjected to flow-sorting using DNA content/ploidy as a parameter. Pan-cytokeratin (pCK) was used to differentiate diploid tumor cell populations from benign diploid stromal cells. DNA has been extracted from these individual tumor cell populations and the mutational profile and copy number status are currently being analyzed using whole exome seqeuencing (WES). Results: We successfully enriched for tumor cells with a purity of higher than 95% using the multi parameter flow-sorting approach. Besides the aneuploid tumors with higher ploidies, we discovered near-diploid tumor populations after enrichment with the pCK antibody. The pCK negative fraction consisting of stromal cells can serve as a germ line control in further downstream analysis as WES. Conclusion: The present study illustrates a unique method that allows high enrichment for tumor cells. Avoiding the dilution of DNA by benign stromal cells, together with the purity of aneuploid tumor cells, the precision of genomic analysis highly increases. At the same time, the diploid and pCK negative stromal cells can serve as a germ line control. Comprehensive genomic profiling of flow-sorted aneuploid and pCK positive diploid tumor populations represent a powerful technique to study intratumoral heterogeneity.