Isolation and detection of circulating tumour cells from metastatic melanoma patients using a slanted spiral microfluidic device

Abstract

Circulating Tumour Cells (CTCs) are promising cancer biomarkers. Several methods have been developed to isolate CTCs from blood samples. However, the isolation of melanoma CTCs is very challenging as a result of their extraordinary heterogeneity, which has hindered their biological and clinical study. Thus, methods that isolate CTCs based on their physical properties, rather than surface marker expression, such as microfluidic devices, are greatly needed in melanoma. Here, we assessed the ability of the slanted spiral microfluidic device to isolate melanoma CTCs via label-free enrichment. We demonstrated that this device yields recovery rates of spiked melanoma cells of over 80% and 55%, after one or two rounds of enrichment, respectively. Concurrently, a two to three log reduction of white blood cells was achieved with one or two rounds of enrichment, respectively. We characterised the isolated CTCs using multimarker flow cytometry, immunocytochemistry and gene expression. The results demonstrated that CTCs from metastatic melanoma patients were highly heterogeneous and commonly expressed stem-like markers such as PAX3 and ABCB5. The implementation of the slanted microfluidic device for melanoma CTC isolation enables further understanding of the biology of melanoma metastasis for biomarker development and to inform future treatment approaches.

Keywords: circulating tumour cells (CTCs); metastatic melanoma; slanted spiral microfluidics.

Figure 1. Recovery rates and purity of melanoma cells isolated using a slanted spiral microfluidic device

Melanoma cells were spiked into blood from healthy donors. (A) Comparison of the mean diameters of melanoma cell lines A2058 and SKMEL5, after five consecutive passages, yielded a statistically significant difference between mean diameters of both cell lines (t-test, P=0.0002). (B) Recovery rates of 5, 10, 50 and 100 CellTracker^™ red-labelled A2058 and SKMEL5 cells spiked in blood from healthy donors after 1X slanted enrichment (n=3). No statistically significant differences were found in the recovery rates for A2058 (P=0.376) and SKMEL5 cell lines (P=0.102) between different spiked amounts. (C) WBC counts in the blood of healthy donors (controls, n=3) and metastatic melanoma patients (n=5) before and after the first and second round of enrichment with the slanted microfluidic device. A one-way ANOVA analysis determined a highly significant depletion of WBCs after the second round of enrichment with the slanted device in both controls (P<0.0001) and patients (P<0.0001). (D) Recovery rates of 50 A2058 and SKMEL5 cells spiked into blood after two rounds of slanted enrichment (controls, n=4). Error bars represent one standard deviation from the mean values across replicates.

Figure 2. Diversity of melanoma CTCs pre and post slanted enrichment

Comparison of phenotype and number of CTCs identified before and after slanted enrichment in 10 metastatic melanoma patients using multimarker flow cytometry. Bars indicate the number of CTCs detected by flow cytometry in each individual, with each phenotype indicated by a colour and pattern.

Figure 3. Gene expression of 5 melanoma-specific genes in samples from healthy controls, spiked samples and CTC fractions from metastatic melanoma patients after slanted enrichment

Heatmap represents the expression levels of the melanoma-associated genes MLANA, TYR, MAGEA, ABCB5 and PAX3, which were assessed by RT-PCR. The 18S rRNA gene was used as an endogenous control. Cellular fractions after slanted processing of the blood from 5 healthy controls were used as negative controls. Spiked samples with 1, 5, 10 and 20 A2058 melanoma cells in a background of 1 × 10⁵ WBCs were used to demonstrate the detection level of this assay. CTC-enriched fractions from 13 metastatic melanomas were interrogated. Spiked samples with 20 A2058 cells were used as positive controls in every run. Each heatmap square corresponds to the reciprocal value of the Ct values for each target gene (1/Ct) for a given sample.

Figure 4. Melanoma-specific multimarker immunofluorescence staining for detection of melanoma CTCs

(A) Optimisation of immunostaining protocol in WBC samples spiked with A2058 cells, cytospun onto glass slides, fixed, permeabilised and immunostained with a combination of cell surface, MCSP (Alexa Fluor® 647, purple), and intracellular -MEL- (gp100, S100 and MLANA; Alexa Fluor® 488, green) markers. DAPI was used for nuclei staining and CD45 (PE, red) as a leukocytic marker. Differential expression of MCSP and MEL markers was observed in A2058 cells. (B) Representative melanoma CTCs detected by this immunofluorescence staining in metastatic melanoma patient blood processed twice through the slanted spiral microfluidic device. Melanoma CTCs had an average cell size of 16 μm (range: 13 – 21 μm). MCSP staining was not detected in any of the isolated melanoma CTCs from patient samples. Scale bar represents size of the cell in μm.