A microfluidic-based filtration system to enrich for bone marrow disseminated tumor cells from breast cancer patients

Abstract

Disseminated tumors cells (DTCs) present in the bone marrow (BM) are believed to be the progenitors of distant metastatic spread, a major cause of mortality in breast cancer patients. To better understand the behavior and therapeutic vulnerabilities of these rare cell populations, unbiased methods for selective cell enrichment are required. In this study, we have evaluated a microfluidic-based filtration system (ParsortixR, Angle PLC), previously demonstrated for use in circulating tumor cell (CTC) capture, to capture BM DTCs. Performance using BM samples was also compared directly to enrichment of CTCs in the peripheral blood (PB) from both metastatic and non-metastatic breast cancer patients. Although the non-specific capture of BM immune cells was significant, the device could routinely achieve significant cytoreduction of BM and PB WBCs and at least 1,000-fold enrichment of DTCs, based on labeled tumor cell spike-in experiments. Detection of previously characterized DTC-associated gene expression biomarkers was greatly enhanced by the enrichment method, as demonstrated by droplet digital PCR assay. Cells eluted from the device were viable and suitable for single cell RNA sequencing experiments. DTCs in enriched BM samples comprised up to 5% of the total cell population, allowing for effective single cell and population-based transcriptional profiling of these rare cells. Use of the Parsortix instrument will be an effective approach to enrich for rare BM DTCs in order to better understand their diverse molecular phenotypes and develop approaches to eradicate these cells to prevent distant disease development in breast cancer patients.

Microfluidic Isolation of Disseminated Tumor Cells from the Bone Marrow of Breast Cancer Patients

“…In our study, an average of 65.6% of spiked cells could be enumerated after the cell separation of BM with Parsortix and subsequent harvesting ( Figure 2 C). These results differ from the 26% yield obtained in another study using DTCs from BM [ 25 ]. However, the results of the present study are in line with the harvest rates of 72.8% of melanoma DTCs in lymph nodes [ 36 ] and 54–69% of CTCs in other studies [ 22 , 23 , 37 ].…”

“…A potential disadvantage of isolating cells based on their size is that BM mononuclear cells are larger in diameter than peripheral blood cells and are therefore also captured during the separation process. This may result in a lower purity of DTCs than what is achieved during the isolation of CTCs, but cell characterization via RNA analysis is still possible [ 25 ]. For single-cell analysis, BM cell depletion steps may be required to bypass DTC identification via staining and morphological characteristics.…”

“…Studies using Parsortix have already been able to successfully enrich and analyze circulating tumor cells (CTCs) from peripheral blood [ 22 , 23 , 24 ]. More recently, it was shown that using Parsortix may also be a feasible method to enrich and characterize DTCs from fresh bone marrow [ 25 ]. Another important feature of Parsortix microfluidic cell separation is that the cells are viable once harvested, theoretically opening the possibility of culturing isolated DTCs.…”

A comparative study of circulating tumor cell isolation and enumeration technologies in lung cancer

“…While the hands-on time is minimal with semi-automated Parsortix® systems, the process of scanning the entire cassette for CTC enumeration could become time-consuming when employing a manual microscope, as discussed later. The harvest option primarily addresses imaging issues encountered with in-cassette staining and is advantageous for subsequent analyses like whole-genome sequencing (WGS) [43], single cell RNA sequencing (scRNAseq) [44, 45], PCR [44, 46, 26], fluorescence in situ hybridization (FISH) [44] and culturing CTCs [47, 48]. Moreover, it is well- suited for analyzing both circulating tumor DNA (ctDNA) and CTCs from the same blood tube [49].…”

Single-cell RNA sequencing of mitotic-arrested prospermatogonia with DAZL::GFP chickens and revealing unique epigenetic reprogramming of chickens

“…Libraries were sequenced with a 2 × 100-bp paired-end protocol on a Novaseq-6000 platform (Illumina, San Diego, CA, USA) to generate at least 40,000 read pairs per cell. The sequencing depth recommended by the manufacturer for the 3′ Gene Expression library is a minimum of 20,000 read pairs per cell, and values in the range of 20,000 to 50,000 read pairs per cell are commonly used in the field [ 37 , 38 ].…”