Microsieves for the detection of circulating tumor cells in leukapheresis product in non-small cell lung cancer patients

Abstract

Background: Circulating tumor cells (CTC) in non-small cell lung cancer (NSCLC) patients are a prognostic and possible therapeutic marker, but have a low frequency of appearance. Diagnostic leukapheresis (DLA) concentrates CTC and mononuclear cells from the blood. We evaluated a protocol using two VyCAP microsieves to filter DLA product of NSCLC patients and enumerate CTC, compared with CellSearch as a gold standard.Methods: DLA was performed in NSCLC patients before starting treatment. DLA product equaling 2×10 8 leukocytes was diluted to 9 mL with CellSearch dilution buffer in a Transfix CTC tube. Within 72 hours the sample was filtered with a 7 μm pore microsieve and subsequently over a 5μm pore microsieve. CTC were defined as nucleated cells which stained for cytokeratin, but lacked CD45 and CD16. CellSearch detected CTC in the same volume of DLA.Results: Of 29 patients a median of 1.4 mL DLA product (range, 0.5-4.1) was filtered (2% of total product) successfully in 93% and 45% of patients using 7 and 5 μm pores, respectively. Two DLA products were unevaluable for CTC detection. Clogging of the 5 μm but not 7 μm microsieves was positively correlated with fixation time (ρ=0.51, P<0.01). VyCAP detected CTC in 44% (12/27) of DLA products.Median CTC count per mL DLA was 0 [interquartile range (IQR): 0-1]. CellSearch detected CTC in 63% of DLA products (median =0.9 CTC per mL DLA, IQR: 0-2.1). CTC counts detected by CellSearch were significantly higher compared with VyCAP (P=0.05).Conclusions: VyCAP microsieves can identify CTC in DLA product, but workflows need to be optimized.

In Vivo Detection of Circulating Cancer-Associated Fibroblasts in Breast Tumor Mouse Xenograft: Impact of Tumor Stroma and Chemotherapy

“…Vimentin failed to show differences between HMFs and cancer cells, regardless of TGFβ stimulation, at both the gene and protein levels, indicating non-specificity (Supplementary Figure S1B). We confirmed that pan-cytokeratin (pan-CK), a widely used biomarker for CTC identification [23], is specifically expressed in MDA-MB-231 breast cancer cells without any signal in HMFs (Figure 1B). Hence, we selected FAP and ITGA5 as biomarkers to detect CAFs and pan-CK as a marker of tumor cells in the following experiment.…”

“…Blood was taken from mice in EDTA tubes, followed by 1% PFA fixation overnight. Then, the blood samples were filtrated through a microsieve platform with a chip pore size of 5 µm (Microsieve, VyCAP) [23]. A similar fixation and permeabilization process was performed, and samples were coincubated with corresponding fluorescently labeled antibodies as mentioned in the Section 2.3, followed by scanning under a Nikon Ti-E automated inverted fluorescence microscope.…”

“…Because most cancer cells originate from epithelial cells, a couple of epithelial biomarkers are exclusively expressed in cancer cells but not in other non-cancer cells in the blood, which facilitates the detection of CTCs from blood samples [17]. For instance, EpCAM (epithelial cell adhesion molecule) and pan-CK (pan-cytokeratin) have been widely used as biomarkers in CTC capture and identification in many studies [18][19][20][21][22][23]. Unlike CTCs, CAFs have multiple sources of origin.…”

Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

“…The high recovery rates obtained for the MetaCell kit in HCT116 and DLD1 cells ( Section 3.2 ) with high cell numbers (~10,000) prompted the investigation of recovery rates using lower CRC cell numbers (10, 100, 500 cells), which is more reflective of the number of CTCs likely to be present in the circulation of CRC patients. The CRC cells from the unfiltered and MetaCell-filtered groups for the different CRC cell numbers were stained with CTC and WBC markers [ 37 ]; cells staining positively for CTC markers and negative for WBC markers were manually counted. For HCT116 cells spiked in blood at 10, 100, and 500, the MetaCell recovery rates were 85.9%, 86.6%, and 87.6%, respectively ( Figure 3 A).…”