Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients

Abstract

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.

Circulating tumour cells in gastrointestinal cancers: food for thought?

“…CTC levels, quantified at baseline and day 28 upon chemotherapy treatment initiation, were also reported to be a prognostic factor in GC, as they were associated with worse progression-free and overall survival [ 28 ]. Of note, CTCs often appear to be heterogeneous and in rare instances, some carcinoma-derived CTCs may even reduce the expression of their epithelial markers [ 29 ]. Therefore, antigen-based isolation using a single marker (e.g EpCAM) may underestimate the phenotypic diversity of CTCs.…”

“…Therefore, antigenbased isolation using a single marker (e.g EpCAM) may underestimate the phenotypic diversity of CTCs. Efforts are made to increase the variety of CTC markers to further improve their characterisation [29]. For example, CD44, cytokeratin, fibroblast growth factor, cell surface vimentin protein, programmed cell death ligand-1, carcinoembryonic antigen, and HER2-positive CTCs were used as potential prognostic markers in GC [30][31][32][33][34].…”

Label-Free Sensing with Metal Nanostructure-Based Surface-Enhanced Raman Spectroscopy for Cancer Diagnosis

“…In this way, these integrated platforms allow miniaturization, automation, sample volume reduction, and efficient sample processing while ensuring high sensitivity, high throughput, and multiplexing. Microfluidics has been coupled with a broad range of sensing technologies such as fluorescence, mass-spectrometry, electrochemistry, and UV–vis. , The diversity of applications arising from these combinations in the context of cancer covers not only diagnostics but also treatment, drug development and dosing, single-cell and genetic material analysis, and organ-on-a-chip applications. − In line with these developments, optofluidic SERS platforms are being increasingly explored in biosensing . Label-free SERS measurements in microfluidic platforms have demonstrated remarkable performance for a broad range of biological targets such as bacteria, − DNA, miRNA, narcotics, and food contaminants. − Very recently, with the advent of personalized medicine and the revolution in data analytics, new opportunities to boost SERS-on-a-chip technologies are being explored. − …”

“… 148 , 149 The diversity of applications arising from these combinations in the context of cancer covers not only diagnostics but also treatment, drug development and dosing, single-cell and genetic material analysis, and organ-on-a-chip applications. 150 − 153 In line with these developments, optofluidic SERS platforms are being increasingly explored in biosensing. 154 Label-free SERS measurements in microfluidic platforms have demonstrated remarkable performance for a broad range of biological targets such as bacteria, 155 − 158 DNA, 159 miRNA, 160 narcotics, 161 and food contaminants.…”

Isolation of acute myeloid leukemia blasts from blood using a microfluidic device

“…13 Recently, microfluidics has been one of the main technologies applied for the isolation and detection of rare cells. Microfluidic devices have been successfully used for sizebased, [14][15][16][17] immune-based, 18,19 or chromatographic-based separation, 20 for cell encapsulation, [21][22][23] and also microfluidic flow cytometry. [24][25][26][27][28] Furthermore, microfluidic devices also offer high efficiency for processing complex fluids with minimal damage to the cells contained in these fluids, even the most sensitive.…”