A Novel CTC-Binding Probe: Enzymatic vs. Shear Stress-Based Detachment Approaches

Abstract

Background/Objectives: Liquid biopsy is a minimally invasive alternative to tissue biopsy and is used to obtain information about a disease from a blood sample or other body fluids. In the context of cancer, circulating tumor cells (CTC) can be used as biomarkers to determine the nature of the tumor, its stage of progression, and the efficiency of the administered therapy through monitoring. However, the low concentration of CTCs in blood (1-10 cells/mL) is a challenge for their isolation. Therefore, a minimally invasive medical device (BMProbe™) was developed that isolates CTCs via antigen-antibody binding directly from the bloodstream. Current investigations focus on the process of detaching bound cells from the BMProbe™ surface for cell cultivation and subsequent drug testing to enable personalized therapy planning. Methods: This article presents two approaches for detaching LNCaP cells from anti-EpCAM coated BMProbes™: enzymatic detachment using TrypLE™ and detachment through enzymatic pretreatment with supplementary flow-induced shear stress. The additional shear stress is intended to increase the detachment efficiency. To determine the flow rate required to gently detach the cells, a computational fluid dynamics (CFD) simulation was carried out. Results: The experimental test results demonstrate that 91% of the bound cells can be detached enzymatically within 10 min. Based on the simulation, a maximum flow rate of 47.76 mL/min was defined in the flow detachment system, causing an average shear stress of 8.4 Pa at the probe edges. The additional flow treatment did not increase the CTC detachment efficiency. Conclusions: It is feasible that the detachment efficiency can be further increased by a longer enzymatic incubation time or higher shear stress. The influence on the integrity and viability of cells must, however, be considered.

Keywords: BMProbe™; TrypLE™; cell detachment; circulating tumor cells (CTC); wall shear stress.

Figure 1

Formation of metastasis: release of cells from the primary tumor, entrance into the bloodstream, and infiltration from the blood vessels into distant tissues. Created with BioRender.com.

Figure 2

BMProbe™ inserted into a peripheral vein via an indwelling cannula.

Figure 3

Imported CAD model created with SolidWorks: geometry of the probe inserted into the tube with defined domains in Star-CCM+.

Figure 4

Test Stands; left: Setup for flow detachment: (1) syringe pump, (2) BMProbe™, (3) stopcock, (4) tube; right: Flow system for attachment of LNCaP cells on the BMProbe™ [38].

Figure 5

Example: LNCaP cells (blue) stained with Hoechst 33342 bound to winding #6 on a BMProbe™; left: before enzymatic detachment, right: after enzymatic detachment.

Figure 6

CFD simulation of wall shear stress on the probe surface using a volume flow of 47.76 mL/min.

Figure 7

Cell counts on the BMProbe™ with conjugated anti-EpCAM before and after treatment. Cell yield: #1: 118, #2: 139, #3: 437, #4: 133, median cell yield: 132.

Figure 8

Detachment efficiencies (DE) when using enzymatic treatment.

Figure 9

Cell counts on the BMProbe™ with conjugated anti-EpCAM before and after treatment. Cell yield: #1: 122, #2: 612, #3: 75, #4: 161, median cell yield: 140.

Figure 10

Detachment efficiencies (DE) when using enzymatic treatment and a flow rate of 47.76 mL/min in the flow detachment system.