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1.
Methods Mol Biol ; 2752: 65-70, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38194028

RESUMO

Tumor heterogeneity has a major role in the development of tumor evasion and resistance to treatments. To study and understand the intrinsic heterogeneity of cancer cells, the use of single-cell isolation technology has had a major boost in recent years, gaining ground to bulk analysis in the study of solid tumors. In the liquid biopsy field, the use of technologies for single-cell analysis has represented a major advance in the study of the heterogeneity of circulating tumor cells (CTCs), providing relevant information about therapy-resistant CTCs. However, single-cell analysis of CTCs is still challenging due to the weakness and scarcity of these cells. In this chapter, we describe a protocol for CTCs isolation at a single-cell level using the VyCAP Puncher system.


Assuntos
Células Neoplásicas Circulantes , Humanos , Separação Celular , Biópsia Líquida , Análise de Célula Única , Tecnologia
2.
Cancers (Basel) ; 13(23)2021 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-34885192

RESUMO

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.

3.
Transl Lung Cancer Res ; 9(4): 1093-1100, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32953488

RESUMO

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.

4.
Lab Chip ; 19(10): 1850-1859, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31041434

RESUMO

Here we describe a combined method to monitor the secretion of molecules produced by single cells, followed by a method to isolate the individual cells that produced these molecules. The method is based on a self-sorting microwell chip that is connected to an activated membrane that collects the produced molecules. The produced molecules are printed by diffusion in small spots onto the membrane. The location of the printed spots can be correlated to the microwell number and the cell that produced these molecules. To demonstrate the method, we used the EpCAM antibody producing hybridoma cell line VU1D9 and a genetically engineered CHO cell-line producing Her2. VU1D9 cells produced 4.6 ± 5.6 pg (mean ± SD) of EpCAM antibody per 24 h and CHO cells 6.5 ± 8.2 pg per 24 h of Herceptin antibody.


Assuntos
Anticorpos/análise , Molécula de Adesão da Célula Epitelial/análise , Análise em Microsséries , Análise de Célula Única , Animais , Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Molécula de Adesão da Célula Epitelial/biossíntese , Molécula de Adesão da Célula Epitelial/imunologia , Humanos , Impressão Tridimensional
5.
Int J Mol Sci ; 20(3)2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30678037

RESUMO

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+, α-EpCAM+, α-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 0⁻5% as compared with CTC counts obtained with the CellSearch® system. Viability of the detected CTC ranged from 0⁻36%. 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.


Assuntos
Separação Celular , Imunofenotipagem , Células Neoplásicas Circulantes/metabolismo , Biomarcadores , Neoplasias da Mama , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Separação Celular/métodos , Sobrevivência Celular , Feminino , Humanos , Imunofenotipagem/métodos , Masculino , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata
6.
Cytometry A ; 93(12): 1255-1259, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30427580

RESUMO

Here we present the Puncher technology for the isolation of single cells. This technology combines a silicon chip with microwells, fluorescence imaging, and a punching method to isolate and transfer the single cells to standard reaction tubes. The technology is compatible with commercially available downstream workflows and instrumentation. Here we focus on the isolation of CTC but the Puncher technology can be applied to isolate single cells from liquid biopsies and more general from cell suspensions. It is especially suited for cell suspensions that contain: Cells of interest at a frequency of 1 per 10,000 or less A low total number of cells ranging from 1 to 100,000, that are present in a volume of 0.01 to 50 mL. The frequency of appearance of CTC in blood is in the order of the 1 per 106 leukocytes. To be able to isolate the single CTC with the Puncher technology, enrichment of the CTC by a 3 logs reduction of the leukocytes is required. Here we describe the use of Rosettesep and Parsortix as examples of pre-enrichment methods that are compatible with the Puncher technology and further downstream applications. © 2018 International Society for Advancement of Cytometry.


Assuntos
Separação Celular/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Humanos , Leucócitos/patologia , Biópsia Líquida/métodos , Técnicas Analíticas Microfluídicas/métodos , Células Neoplásicas Circulantes/patologia
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