and J.R. by functionalizing the membrane with specific antibodies against a CTC-characteristic surface protein such as the epithelial cell adhesion molecule (EpCAM). A common shortcoming of both filtration approaches is that there is still a poor understanding of the enrichment process and the systems developed so far are frequently managed under non-optimized conditions. To address this, systematic filtration experiments are performed with this work using the EpCAM+ cell collection MCF-7 as CTC-model and standard track-etched membranes altered with or without antibodies against EpCAM. The influences of the key filtration parameters time and applied pressure are analyzed and it is found that in all cases the degree of cell recovery is limited by a lysis process which occurs within the membrane surface. Counterintuitively, it is found that filtration at rather high pressures is definitely advantageous to make sure high recovery rates. To describe the pressure-induced lysis process a GDC-0449 (Vismodegib) biophysical model is definitely developed. This model allows the dedication of optimum filtration conditions to accomplish both high malignancy cell recovery and large blood sample throughput. It is demonstrated that this way practically 100% of spiked malignancy cells can be recovered from milliliters of undiluted whole blood within seconds. Intro GDC-0449 (Vismodegib) Malignancy is definitely a major cause of death and morbidity worldwide1. In Europe only, there were an estimated 3.45 million new cases of cancer and 1.75 million deaths from cancer in 20122. Including the most common types of malignancy like breast, colorectal, prostate and lung cancer, carcinomas represent by far the most abundant class of tumors. Carcinomas are solid tumors derived from epithelial cells and most deaths from this class of tumors are caused by the haematogenous spread of malignancy cells from the primary tumor into distant organs and their subsequent growth to metastases3,4. With this complex process Circulating tumor cells (CTCs) were found to play a key role with this complex process?and their reliable detection and characterization could enable new and effective strategies for cancer diagnosis, monitoring and treatment4C7. However, the analysis of CTCs still represents a strong analytical challenge because of the ultra-low abundance of a few cells per mL of blood, while they are at the same time covered by billions of blood cells7C9. The reliable analysis of CTCs using standard microscopic methods like immunocytochemistry (ICC), consequently, strongly depends on an efficient CTC-enrichment step. It was found out already in 1964 by S. H. Seal that CTCs can be isolated from whole blood by classical dead-end microfiltration because of the larger size and lower deformability compared to normal blood cells10. Compared to other methods of cell separation, filtration is of unique interest due to its high effectiveness, cost-effectiveness, handling-simplicity, good compatibility with downstream analysis of cells and high sample throughput7,11C15. Filtration studies performed so far, primarily focused on different membrane geometries16C25, or were concerned with the assessment of filtration to other methods of enrichment26,27. With the aim to enhance the overall performance of classical filtration, the concept of affinity filtration was launched28. The key concept of the system is definitely to enrich CTCs ARHGEF11 based on a combination of mechanical and molecular connection with the membrane and therefore include a second level of selectivity through immobilization of CTC-selective capture molecules (e.g., anti-EpCAM) within the membrane. This concept has in GDC-0449 (Vismodegib) the meantime been analyzed by other investigators and a beneficial effect of the affinity filtration has been reported29. However, there has not yet been a systematic study within the influence of the filtration parameters within the enrichment process. This leaves us having a still poor understanding of the filtration process and the systems developed so far operate under non-optimized conditions. The aim of this work is definitely to address these shortcomings.
