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

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 2×108 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

Measurement of the drug sensitivity of single prostate cancer cells

The treatment of cancer faces a serious challenge as cancer cells within patients are heterogeneous and frequently resistant to therapeutic drugs. Here, we introduce a technology enabling the assessment of single cancer cells exposed to different drugs. PCa cells were individually sorted in self-seeding microwells, cultured for 24 h, and then exposed to several drugs to induce (R1881) or inhibit (Enzalutamide/Abiraterone) the secretion of a protein (PSA). Cell viability and PSA secretion of each individual prostate cell were monitored over a 3-day period. The PSA protein secreted by each cell was captured on a PVDF membrane through a pore in the bottom of each well. The basal PSA secretion was found to be 6.1 ± 4.5 and 3.7 ± 1.9 pg/cell/day for LNCaP and VCaP, respectively. After exposure to R1881, the PSA secretion increased by ~90% on average and was not altered for ~10% of the cells. PSA production decreased in the majority of cells after exposure to enzalutamide and abiraterone

Self-Seeding Microwells to Isolate and Assess the Viability of Single Circulating Tumor Cells

The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, &#945;-EpCAM+, &#945;-CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0&#8315;5% as compared with CTC counts obtained with the CellSearch&#174; system. Viability of the detected CTC ranged from 0&#8315;36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses