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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE118478 | GEO | 2018/08/14

REPOSITORIES: GEO

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Project description:Cutaneous melanoma first metastasizes into sentinel lymph nodes that control the lymphatic drain from the area of the primary tumor. This observation is used clinically for melanoma patients with primary melanomas thicker than 1mm (tumor stage ≥T2a), for these patients sentinel lymph node biopsy has become an important and routinely performed diagnostic procedure. The importance of sentinel node analysis is reflected by a significant better prognosis of melanoma patients with tumor free sentinel nodes compared to patients with metastatic sentinel nodes. Although intensively studied, not much is known about mechanisms responsible for the development of melanoma metastasis.To analyze gene expression in mouse SLNs of M24met tumor bearing animals as compared to tumor free control animals, SLNs were taken at different time points and analyzed for the presence of human M24met to classify SLNs into control, negative, macro metastatic SLN. After categorized SLNs were subjected to microarray analysis. To analyze gene expression in mouse SLNs of human M24met tumor bearing animals as compared to tumor free control animals, SLNs were taken at different time points and analyzed for the presence of human M24met to classify SLNs into control, negative, macro metastatic SLN. Briefly, explanted SLNs were stored in RNA later (Ambion, Austin, TX) and DNA as well as RNA was extracted using AllPrep DNA/RNA Mini Kit and RNeasy Micro Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. To detect human cells in mouse SLNs we used a polymerase chain reaction method for the detection of a human-specific 850-bp fragment of the alpha-satellite DNA on human chromosome 17. For analysis of gene expression RNA from control SLN from tumor free animals (control), RNA from tumor negative SLN (negative), and RNA from macro metastatic SLN (positive) from tumor bearing animals was used to analyze gene expression on Mouse Genome 430A 2.0 Arrays (Affymetrix, Santa Clara, CA)

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

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