| Popis: |
In breast cancer research, immunomagnetic enrichment of circulating tumor cells (CTCs) from body fluids has impressively evolved over the last decades. However, there is growing interest in further singularizing these pre-enriched rare cells to decrease signal-to-noise ratio for downstream molecular analysis, e.g., to distinguish between hormone receptor-associated tumor subtypes. This can be done by a combinatory principle to link magnetic cell separation with flow cytometry and single cell dispensing. We have recently introduced an automated benchtop platform with a microfluidic disposable cartridge to immunomagnetically enrich, fluorescence-based detect and dispense single cells from biological samples. Herein, we showcase the fine-tuning of microfluidic cell isolation in dependency of bead-binding on the cell surface. We implemented a gating function for the cytometer subunit of the benchtop platform to selectively dispense cells instead of autofluorescent objects. Finally, we developed a simplified qPCR protocol without RNA purification targeting breast cancer-relevant progesterone and estrogen receptor, Muc-1, Her-2, EpCAM and CXCR4 transcripts. In conclusion, the presented results markedly demonstrate a future diagnostic and therapy-accompanying semi-automated workflow using immunomagnetic enrichment, fluorescence-based isolation and dispensing of circulating tumor cells to achieve tumor subtyping by means of rapid, simple and immediate molecular biological examination of single cells. |
