Microfluidic enrichment, isolation and characterization of disseminated melanoma cells from lymph node samples

For the first time in melanoma, novel therapies have recently shown efficacy in the adjuvant therapy setting, which makes companion diagnostics to guide treatment decisions a desideratum. Early spread of disseminated cancer cells (DCC) to sentinel lymph nodes (SLN) is indicative of poor prognosis in melanoma and early DCCs could therefore provide important information about the malignant seed. Here, we present a strategy for enrichment of DCCs from SLN suspensions using a microfluidic device (ParsortixTM, Angle plc). This approach enables the detection and isolation of viable DCCs, followed by molecular analysis and identification of genetic changes. By optimizing the workflow, the established protocol allows a high recovery of DCC from melanoma patient-derived LN suspensions with harvest rates above 60%. We then assessed the integrity of the transcriptome and genome of individual, isolated DCCs. In LNs of melanoma patients, we detected the expression of melanoma-associated transcripts including MLANA (encoding for MelanA protein), analysed the BRAF and NRAS mutational status and confirmed the malignant origin of isolated melanoma DCCs by comparative genomic hybridization. We demonstrate the feasibility of epitope-independent isolation of LN DCCs using ParsortixTM for subsequent molecular characterization of isolated single DCCs with ample application fields including the use for companion diagnostics or subsequent cellular studies in personalized medicine. The analysis aimed to indentify profiles of copy number changes in genomic DNA of disseminated cancer cells (DCCs) in lymph nodes (LNs). For this, DCCs were enriched using the ParsortixTM system. After harvest, cells were collected and transferred on chamber slides, enabling manual isolation of single cells using a micromanipulator. For further molecular downstream analysis, isolated single cells were subjected to simultaneous whole transcriptome amplification (WTA) and whole genome amplification (WGA). Subsequently, single-cell DNA was amplified using the Ampli1 WGA Kits and subjected to single-cell aCGH analysis according to previously published protocol (Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907). The analysis included 4 single DCCs obtained from 2 melanoma patients. The reference sample used for all aCGH experiments consisted of a pool of four single-cell WGA products generated from WBCs of a healthy male donor.