Circulating tumor cell investigation in breast cancer patient-derived xenograft models by automated immunofluorescence staining, image acquisition, and single cell retrieval ... View Full Text


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Article Info

DATE

2019-12

AUTHORS

Arturo B. Ramirez, Raksha Bhat, Debashish Sahay, Carmine De Angelis, Hariprasad Thangavel, Sina Hedayatpour, Lacey E. Dobrolecki, Agostina Nardone, Mario Giuliano, Chandandeep Nagi, Mothaffar Rimawi, C. Kent Osborne, Michael T. Lewis, Jackie L. Stilwell, Eric P. Kaldjian, Rachel Schiff, Meghana V. Trivedi

ABSTRACT

BACKGROUND: Breast cancer patient-derived xenograft (BC-PDX) models represent a continuous and reproducible source of circulating tumor cells (CTCs) for studying their role in tumor biology and metastasis. We have previously shown the utility of BC-PDX models in the study of CTCs by immunohistochemistry (IHC) on serial paraffin sections and manual microscopic identification of cytokeratin-positive cells, a method that is both low-throughput and labor-intensive. We therefore aimed to identify and characterize CTCs from small volume mouse blood samples and examined its practical workflow in a study of BC-PDX mice treated with chemotherapy using an automated imaging platform, the AccuCyte®-CyteFinder® system. METHODS: CTC analysis was conducted using blood from non-tumor bearing SCID/Beige mice spiked with human breast cancer cells, BC-PDX-bearing mice, and BC-PDX mice treated with vehicle or chemotherapeutic agent(s). After red blood cell lysis, nucleated cells were mixed with transfer solution, processed onto microscope slides, and stained by immunofluorescence. The CyteFinder automated scanning microscope was used to identify CTCs, defined as nucleated cells that were human cytokeratin-positive, and mouse CD45-negative. Disaggregated primary BC-PDX tumors and lung metastatic nodules were processed using the same immunostaining protocol. Collective expression of breast cancer cell surface markers (EpCAM, EGFR, and HER2) using a cocktail of target-specific antibodies was assessed. CTCs and disaggregated tumor cells were individually retrieved from slides using the CytePicker® module for sequence analysis of a BC-PDX tumor-specific PIK3CA mutation. RESULTS: The recovery rate of human cancer cells spiked into murine blood was 83 ± 12%. CTC detection was not significantly different from the IHC method. One-third of CTCs did not stain positive for cell surface markers. A PIK3CA T1035A mutation present in a BC-PDX tumor was confirmed in isolated single CTCs and cells from dissociated metastatic nodules after whole genome amplification and sequencing. CTC evaluation could be simply implemented into a preclinical PDX therapeutic study setting with substantial improvements in workflow over the IHC method. CONCLUSIONS: Analysis of small volume blood samples from BC-PDX-bearing mice using the AccuCyte-CyteFinder system allows investigation of the role of CTCs in tumor biology and metastasis independent of surface marker expression. More... »

PAGES

220

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12885-019-5382-1

DOI

http://dx.doi.org/10.1186/s12885-019-5382-1

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1112768710

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/30871481


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curl -H 'Accept: application/n-triples' 'https://scigraph.springernature.com/pub.10.1186/s12885-019-5382-1'

Turtle is a human-readable linked data format.

curl -H 'Accept: text/turtle' 'https://scigraph.springernature.com/pub.10.1186/s12885-019-5382-1'

RDF/XML is a standard XML format for linked data.

curl -H 'Accept: application/rdf+xml' 'https://scigraph.springernature.com/pub.10.1186/s12885-019-5382-1'


 

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