The Integration of a Three-Dimensional Spheroid Cell Culture Operation in a Circulating Tumor Cell (CTC) Isolation and Purification Process: A Preliminary Study of the Clinical Significance and Prognostic Role of the CTCs Isolated from the Blood Samples of Head and Neck Cancer Patients

Abstract

Conventional positive and negative selection-based circulating tumor cell (CTC) isolation methods might generally ignore metastasis-relevant CTCs that underwent epithelial-to- mesenchymal transition and suffer from a low CTC purity problem, respectively. To address these issues, we previously proposed a 2-step CTC isolation method integrating a negative selection CTC isolation and subsequent spheroid cell culture. In addition to its ability to isolate CTCs, more importantly, the spheroid cell culture used could serve as a cell culture model mimicking the process of new tumor tissue formation during cancer metastasis. Therefore, it is promising not only to selectively isolate metastasis-relevant CTCs but also to test the potential of cancer metastasis and thus the prognosis of disease. To explore these issues, experiments were performed. The key findings of this study demonstrated that the method was able to harvest both epithelial (E)- and mesenchymal (M)-type CTCs without selection bias. Moreover, both the M-type CTC count and the information obtained from the multidrug resistance-associated protein 2 (MRP2) and MRP5 gene expression analysis of the CTCs isolated via the 2-step CTC isolation method might be able to serve as prognostic factors for progression-free survival in head and neck squamous cell carcinoma.

Enhancing Prediction Performance by Add-On Combining Circulating Tumor Cell Count, CD45neg EpCAMneg Cell Count on Colorectal Cancer, Advance, and Metastasis

“…Based on the proposed negative selection-based protocol for cell enumeration [ 26 ], the concentration of CD45 neg EpCAM pos CTCs and CD45 neg EpCAM neg cells in a blood sample was proven to be able to significantly discriminate between healthy donors and patients with any stage of colorectal cancer ( Figure 2 ), even though the pathological stage of many enrolled CRC patients was early (stages 0–II) ( Figure 4 A,C). This method of CTC counting has the limitations of (1) background cell contamination when CTC is extremely rare; (2) incomplete or oversaturated antibody staining resulting in underestimated or overestimated cell counting.…”

“…Bioscience, San Diego, CA, USA) at 4 °C. Collection was quickly followed by blood sample processing using the negative selection-based cell enrichment scheme, reported on previously [ 26 ], within 24 h so ass to avoid the alteration of the gene and surface protein expression [ 27 ]. Briefly, the blood sample was first mixed with an erythrocyte lysis buffer (150 mM NH 4 Cl, 3.5 mM KHCO 3 , and 50 μM EDTA (pH 7.0)) and was then incubated for 8 min.…”

“…Based on the above discussions, overall, one question is raised: can the use of any combination of these cellular or molecular biomarkers (i.e., the CD45 neg EpCAM pos CTC count, CD45 neg EpCAM neg cell count, or CEA level) improve the predictive performance for CRC disease (e.g., earlier diagnosis) or its status (e.g., advanced or metastatic CRC)? To answer this question, a prospective translational trial was carried out in which a negative selection-based CTC enumeration technique [ 26 ] was utilized to quantify the two cell populations as described previously in the blood samples of healthy blood donors and CRC patients with different stages of the disease. Therefore, our study aimed to combine CD45 neg EpCAM pos CTCs and CD45 neg EpCAM neg cells to predict CRC, besides, based on existing conventional serum biomarker CEA, to evaluate add-on combining both CTC molecular biomarkers whether can improve the performance of AUROC for advanced CRC and CRC metastasis prediction.…”

Liquid Biopsies in Head and Neck Cancer: Current State and Future Challenges

“…Label-free inertial microfluidics approaches, for example, can avoid underestimation of CTC exhibiting downregulation of surface marker expression, and have been reported to be able to detect a larger CTC pool (3–133 CTC/mL of blood) than what had been previously achieved by the CellSearch system [ 21 ]. Researchers have also developed a 2-step CTC isolation and purification method, integrating a negative selection-based CTC isolation scheme and a subsequent 8-day spheroid cell culture for the further purification of CTCs, thereby isolating CTCs with higher purity [ 22 ].…”

Evaluation of Human Mammaglobin as a Biomarker of Circulating Tumor Cells in Breast Cancer and Clinical Verification

“…Cytokeratin 19 (CK19) CK19 is a typical marker of epithelial cells and CTCs. However, the level of CK19 expression for those CTCs undergo the Epithelial-mesenchymal transition (EMT) process will present low expression level of CK19 [20,21], inevitably brings false negative results [22].Human mammaglobin (hMAM) are tissue-speci c markers of breast epithelial tissue and their abnormal expression is closely related to micrometastases [23,24], hMAM is expressed only in mammary epithelial cells and overexpressed mainly in breast cancer cells [25,26].…”