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In recent years, avidity has emerged as a critical parameter in antibody design, yet most current analytical instruments are limited to measuring affinity alone. This study aims to evaluate the capabilities and advantages of a novel surface plasmon resonance imaging instrument, CellVysion, designed to quantify cell–antibody avidity using a continuous antibody density gradient. A key feature of this approach is the identification of a “tipping point”—the specific ligand density, measured in µRIUs, at which cells remain bound to the sensor surface under defined shear flow conditions. In this paper, we present the technical principles and application of this method, demonstrating how avidity can be quantitatively assessed across different antibody–cell line combinations.

When evaluating EpCAM-based enrichment technologies for circulating tumour cells (CTCs), the cell lines used should closely resemble real CTCs, meaning the EpCAM expression of CTCs needs to be known, but also the EpCAM expression of cell lines at different institutions and times is important. As the number of CTCs in the blood is low, we enriched CTCs through the depletion of leukocytes from diagnostic leukapheresis products of 13 prostate cancer patients and measured EpCAM expression using quantitative flow cytometry. Antigen expression was compared between multiple institutions by measuring cultures from each institution. Capture efficiency was also measured for one of the used cell lines. Results show CTCs derived from castration-sensitive prostate cancer patients have varying but relatively low EpCAM expression, with median expression per patient ranging from 35 to 89,534 (mean 24,993) molecules per cell. A large variation in the antigen expression of identical cell lines cultured at different institutions was found, resulting in recoveries when using the CellSearch system ranging from 12 up to 83% for the same cell line. We conclude that large differences in capture efficiency can occur while using the same cell line. To closely resemble real CTCs from castration-sensitive prostate cancer patients, a cell line with a relatively low EpCAM expression should be used, and its expression should be monitored frequently.

BackgroundCirculating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes.MethodsPatients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 108 leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs.ResultsFifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p < 0.01). CTCs per 7.5 mL DLA product were median 9.2 times (interquartile range = 5.6–24.0) higher than CTCs in 7.5 mL blood. RosetteSEP did not significantly improve CTC detection (pretreatment: 34/55, 62%, post treatment: 16/34, 47%) and CTCs per mL even decreased compared to DLA (p = 0.04)..Patients with advanced-stage disease with DLA-CTC after treatment showed fewer tumour responses and shorter progression-free survival (PFS) than those without DLA-CTC (median PFS, 2.0 vs 12.0 months, p < 0.01). DLA-CTC persistence after treatment was independent of clinical factors associated with shorter PFS (hazard ratio (HR) = 5.8, 95% confidence interval (CI), 1.4–35.5, p = 0.02). All evaluable CTCs showed aneuploidy.ConclusionsDLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS.Trial registrationThe study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).

The clinical utility of circulating tumor cells (CTCs) is hampered by the low number of cells detected. Diagnostic leukapheresis (DLA) offers a solution but, due to the observed non‐specific binding and clumping, processing of DLA samples using the CellSearch system only allows for the processing of aliquots consisting of ~ 2% of the total DLA sample per test. Here, we introduce a flow enrichment target capture Halbach‐array (FETCH)‐based separation method in combination with a DNase preprocessing step to capture CTCs from larger fractions of DLA products without clumping. To evaluate the FETCH method, we processed peripheral blood samples from 19 metastatic castration‐naïve prostate cancer (mCNPC) patients with CellSearch, and processed 2% aliquots of leukapheresis samples from the same patients with CellSearch as well as FETCH with or without DNase preprocessing. Using 2% aliquots from six patients, the use of FETCH with fewer immunomagnetic epithelial cellular adhesion molecule (EpCAM) conjugated ferrofluids was tested, whereas 20% aliquots from four patients were used to evaluate the processing of 10‐fold larger DLA samples using FETCH. Results show that the cell clumping normally seen after immunomagnetic enrichment of DLA material was greatly reduced with the use of DNase pretreatment, while the number of CTCs detected was not affected. The number of CTCs detected in 2% aliquots of DLA using FETCH was unchanged compared to CellSearch and did not decrease when using down to 10% of the volume of immunomagnetic anti‐EpCAM ferrofluids normally used in a CellSearch test, whereas the number of co‐enriched white blood cells reduced a median 3.2‐fold. Processing of a 20% aliquot of DLA with FETCH resulted in a 14‐fold increase in CTCs compared to the processing of 2% aliquots of DLA using CellSearch and a total 42‐fold median increase in CTCs compared to peripheral‐blood CellSearch. The number of circulating tumor cells obtained from prostate cancer patients was increased approximately 5‐fold compared to regular CellSearch when processing 2 mL diagnostic leukapheresis material aliquots and increased by 44‐fold when processing 20 mL DLA aliquots using the flow enrichment target capture Halbach‐array. Clumping of cells during the procedure could be prevented by pre‐incubation with DNase.

Due to the low frequency of circulating tumor cells (CTC), the standard CellSearch method of enumeration and isolation using a single tube of blood is insufficient to measure treatment effects consistently, or to steer personalized therapy. Using diagnostic leukapheresis this sample size can be increased; however, this also calls for a suitable new method to process larger sample inputs. In order to achieve this, we have optimized the immunomagnetic enrichment process using a flow-through magnetophoretic system. An overview of the major forces involved in magnetophoretic separation is provided and the model used for optimizing the magnetic configuration in flow through immunomagnetic enrichment is presented. The optimal Halbach array element size was calculated and both optimal and non-optimal arrays were built and tested using anti-EpCAM ferrofluid in combination with cell lines of varying EpCAM antigen expression. Experimentally measured distributions of the magnetic moment of the cell lines used for comparison were combined with predicted recoveries and fit to the experimental data. Resulting predictions agree with measured data within measurement uncertainty. The presented method can be used not only to optimize magnetophoretic separation using a variety of flow configurations but could also be adapted to optimize other (static) magnetic separation techniques.

Human aging is associated with loss of function and regenerative capacity. Human bone marrow derived mesenchymal stromal cells (hMSCs) are involved in tissue regeneration, evidenced by their capacity to differentiate into several lineages and therefore are considered the golden standard for cell-based regeneration therapy. Tissue maintenance and regeneration is dependent on stem cells and declines with age and aging is thought to influence therapeutic efficacy, therefore, more insight in the process of aging of hMSCs is of high interest. We, therefore, hypothesized that hMSCs might reflect signs of aging. In order to find markers for donor age, early passage hMSCs were isolated from bone marrow of 61 donors, with ages varying from 17-84, and clinical parameters, in vitro characteristics and microarray analysis were assessed. Although clinical parameters and in vitro performance did not yield reliable markers for aging since large donor variations were present, genome-wide microarray analysis resulted in a considerable list of genes correlating with human age. By comparing the transcriptional profile of aging in human with the one from rat, we discovered follistatin as a common marker for aging in both species. The gene signature presented here could be a useful tool for drug testing to rejuvenate hMSCs or for the selection of more potent, hMSCs for cell-based therapy.

Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix mainly composed of collagen type I. Here we assessed the potential role of endogenous collagen synthesis in hMSC differentiation and stem cell maintenance. We observed a sharp reduction in proliferation rate of hMSCs in the absence of ascorbic acid, concomitant with a reduction in osteogenesis in vitro and bone formation in vivo. In line with a positive role for collagen type I in osteogenesis, gene expression profiling of hMSCs cultured in the absence of ascorbic acid demonstrated increased expression of genes involved in adipogenesis and chondrogenesis and a reduction in expression of osteogenic genes. We also observed that matrix remodeling and anti-osteoclastogenic signals were high in the presence of ascorbic acid. The presence of collagen type I during the expansion phase of hMSCs did not affect their osteogenic and adipogenic differentiation potential. In conclusion, the collagenous matrix supports both proliferation and differentiation of osteogenic hMSCs but, on the other hand, presents signals stimulating matrix remodeling and inhibiting osteoclastogenesis.

Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

The clinical implementation of circulating tumor cells (CTCs) as a predictive tool for therapy efficacy in the growing field of precision oncology likely requires advanced CTC phenotyping. While their predictive value for disease progression and therapy response has extensively been shown, the low number of CTCs that are obtained from whole blood tubes limits their clinical application. The magnetic enrichment of CTCs using the FDA-cleared CellSearch system is limited by the expression of the epithelial cell adhesion molecule (EpCAM) on CTC, and an increase in CTC yield may be obtained through enhanced capture of low EpCAM expressing cells. In this study, we compared the Flow Enabled Target Capture Halbach (FETCH) magnetic enrichment technology with the FDA-cleared CellSearch system for CTC capture using paired blood samples from 34 patients with non-small-cell lung, breast, and prostate cancer. Results show a statistically significant median 1.5-fold increase in CTCs captured with the FETCH system compared with CellSearch, accompanied by a median twofold reduction in non-specifically captured leukocytes. This increase in CTC capture can aid the comprehensive analysis of CTCs in a larger number of patients, further supporting clinical implementation of CTC-based diagnostics.

Circulating tumor cells (CTCs) are important biomarkers for cancer diagnosis and treatment monitoring. However, their scarcity limits their utility as current enrichment techniques are hampered by low volume throughput and/or the inability to capture CTCs with low target antigen densities. Our group previously reported a device capable of processing samples in a flow manner using an optimized Halbach array to enhance the capture of low EpCAM-expressing cells (Flow-through Immunomagnetic CTC Enrichment system). In this study, we tested the capture efficiency of eight commercially available streptavidin magnetic beads using this device to identify the most suitable bead. Results indicate that using this system, the best-performing magnetic beads are in the ~ 100 to ~ 150 nm size range. Considering the combination of binding efficiency and final sample purity, we found that among the beads tested in combination with biotinylated anti-EpCAM, MojoSort Streptavidin Nanobeads performed the best, with high capture efficiencies for both the high EpCAM expressing LNCaP and low EpCAM expressing PC3-9 cell lines. For CTC enrichment from the blood of cancer patients, reducing the number of WBCs co-enriched with these beads will be essential, especially when processing large-volume samples acquired, for instance, through diagnostic leukapheresis to overcome the limitations caused by the scarcity of CTCs.