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. 2023 Sep 11;24(18):13930.
doi: 10.3390/ijms241813930.

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

Léa L Volmer  1   2 , Cansu E Önder  1 , Barbara Volz  1 , Anjali R Singh  1 , Sara Y Brucker  2 , Tobias Engler  2 , Andreas D Hartkopf  1   2 , André Koch  1
Affiliations

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

Léa L Volmer et al. Int J Mol Sci. .

Abstract

Disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients are putative precursors of metastatic disease, and their presence is associated with an adverse clinical outcome. To achieve the personalization of therapy on a clinical routine level, the characterization of DTCs and in vitro drug testing on DTCs are of great interest. Therefore, biobanking methods, as well as novel approaches to DTC isolation, need to be developed. In this study, we established a protocol for the biobanking of BM samples and evaluated a microfluidic-based separation system (Parsortix®) for the enrichment of cryopreserved DTCs. We were able to successfully isolate viable DTCs after the prior cryopreservation of BM samples. We calculated a significant increase of up to 90-fold in harvested DTCs with the proposed method compared to the current standard techniques, opening up new analysis possibilities for DTCs. Our advanced method further presents options for 3D DTC cultures, enabling the individualized testing of targeted therapies for BC patients. In conclusion, we present a novel approach for DTC enrichment, with possibilities for future clinical implications.

Keywords: Parsortix; breast cancer; disseminated tumor cells (DTCs); liquid biopsy; microfluidic cell separation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Spiking experiments with Ficoll gradient enrichment of PBMC. (A) Workflow of experiment. SK-BR-3 cells (Cell Tracker Green labeled) were spiked in whole BM followed by PBMC isolation. The PBMC fraction was then subjected to either cytospinning or Parsortix enrichment. CTG-positive cells were then counted on cytospins (B) or in the Parsortix cassette (C). (B) Box plot for enumerated CTG-positive cells on cytospins normalized to the total number of spiked cells (n = 6). (C) Box plot for enumerated CTG-positive cells present in the Parsortix harvest cassette normalized to the total number of spiked cells (n = 4).
Figure 2
Figure 2
Spiking experiments with microfluidic separation of whole BM. (A) Workflow of experiment. SK-BR-3 cells (Cell Tracker Green labeled) were spiked in whole BM followed by Parsortix enrichment. Cells were then enumerated before harvest (B) and after harvest (C). (B) Box plot of CTG-positive cells captured in the Parsortix cassette normalized to the total number of spiked cells. (C) Box plot of CTG-positive cells (normalized to the total number of spiked cells) recovered from whole BM after Parsortix enrichment. A total of eight experiments were performed.
Figure 3
Figure 3
Recovery of patient DTCs from cryopreserved BM. (A) Workflow of experiment; BM samples were cryopreserved and subsequently thawed for Parsortix enrichment (see Methods for details). (B) Immunofluorescence images of harvested cells. DTCs can be identified as pan-CK+/DAPI+, and mononuclear cells can be identified as pan-CK-/DAPI+.
Figure 4
Figure 4
Cell separation with Parsortix enables 3D culture of SK-BR-3 cells after BM cryopreservation. Brightfield images (days 0, 7, and 11 of culture) of SK-BR-3 cells (stably expressing GFP) cultured in BME after spiking into BM sample, cryopreservation, and separation with Parsortix. Insert (day 11) shows a grape-like organoid as brightfield and GFP. Scale bar: 300 μm.
See this image and copyright information in PMC

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