Dataset Information

ALCAM: A Novel Surface Marker on EpCAM^low Circulating Tumor Cells.

ABSTRACT: Background: Current strategies in circulating tumor cell (CTC) isolation in pancreatic cancer heavily rely on the EpCAM and cytokeratin cell status. EpCAM is generally not considered a good marker given its transitory change during Epithelial to Mesenchymal Transition (EMT) or reverse EMT. There is a need to identify other surface markers to capture the complete repertoire of PDAC CTCs. The primary objective of the study is to characterize alternate surface biomarkers to EpCAM on CTCs that express low or negligible levels of surface EpCAM in pancreatic cancer patients. Methods: Flow cytometry and surface mass spectrometry were used to identify proteins expressed on the surface of PDAC CTCs in culture. CTCs were grown under conditions of attachment and in co-culture with naïve neutrophils. Putative biomarkers were then validated in GEMMs and patient samples. Results: Surface proteomic profiling of CTCs identified several novel protein biomarkers. ALCAM was identified as a novel robust marker in GEMM models and in patient samples. Conclusions: We identified several novel surface biomarkers on CTCs expressed under differing conditions of culture. ALCAM was validated and identified as a novel alternate surface marker on EpCAM^low CTCs.

SUBMITTER: Signorelli R

PROVIDER: S-EPMC9405826 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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ALCAM: A Novel Surface Marker on EpCAM<sup>low</sup> Circulating Tumor Cells.

Signorelli Rossana R Giret Teresa Maidana TM Umland Oliver O Hadisurya Marco M Lavania Shweta S Charles Richard John Lalith JL Middleton Ashley A Boone Melinda Minucci MM Ergonul Ayse Burcu AB Tao Weiguo Andy WA Amirian Haleh H Iliuk Anton A Khan Aliya A Diaz Robert R Cortes Daniel Bilbao DB Garcia-Buitrago Monica M Charles Jacob Harrys Kishore HK

Biomedicines 20220816 8

<b>Background:</b> Current strategies in circulating tumor cell (CTC) isolation in pancreatic cancer heavily rely on the EpCAM and cytokeratin cell status. EpCAM is generally not considered a good marker given its transitory change during Epithelial to Mesenchymal Transition (EMT) or reverse EMT. There is a need to identify other surface markers to capture the complete repertoire of PDAC CTCs. The primary objective of the study is to characterize alternate surface biomarkers to EpCAM on CTCs tha ...[more]

PMID: 36009530

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Project description:Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Project description:Circulating tumor cells (CTCs) have substantial clinical implications in cancer diagnosis and monitoring. Although significant progress has been made in developing technologies for CTC detection and counting, the ability to quantitatively detect multiple surface protein markers on individual tumor cells remains very limited. In this work, we report a multiplexed method that uses magnetic multicolor surface-enhanced Raman scattering (SERS) nanotags in conjunction with a chip-based immunomagnetic separation to quantitatively and simultaneously detect four surface protein markers on individual tumor cells in whole blood. Four-color SERS nanotags were prepared using magnetic-optical iron oxide-gold core-shell nanoparticles with different Raman reporters to recognize four different cancer markers with respective antibodies. A microfluidic device was fabricated to magnetically capture the nanoparticle-bound tumor cells and to perform online negative staining and single-cell optical detection. The level of each targeted protein was obtained by signal deconvolution of the mixed SERS signals from individual tumor cells using the classic least squares regression method. The method was tested with spiked tumor cells in human whole blood with three different breast cancer cell lines and compared with the results of purified cancer cells suspended in a phosphate buffer solution. The method, with either spiked cancer cells in blood or purified cancer cells, showed a strong correlation with purified cancer cells by enzyme-linked immunosorbent assay, suggesting the potential of our method for the reliable detection of multiple surface markers on CTCs. Combining immunomagnetic enrichment with high specificity, multiplexed targeting for the capture of CTC subpopulations, multicolor SERS detection with high sensitivity and specificity, microfluidics for handling rare cells and magnetic-plasmonic nanoparticles for dual enrichment and detection, our method provides an integrated, yet a simple and an efficient platform that has the potential to more sensitively detect and monitor cancer metastasis.

Dataset Information

ALCAM: A Novel Surface Marker on EpCAM^low Circulating Tumor Cells.

Publications

ALCAM: A Novel Surface Marker on EpCAM<sup>low</sup> Circulating Tumor Cells.

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Dataset Information

ALCAM: A Novel Surface Marker on EpCAMlow Circulating Tumor Cells.

Publications

ALCAM: A Novel Surface Marker on EpCAM<sup>low</sup> Circulating Tumor Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

ALCAM: A Novel Surface Marker on EpCAM^low Circulating Tumor Cells.