A Laboratory-Friendly CTC Identification: Comparable Double-Immunocytochemistry with Triple-Immunofluorescence

SIMPLE SUMMARY: Tumor cells that circulate in the peripheral blood of patients with solid tumors are called circulating tumor cells. Since the source of circulating tumor cells are from primary cancer sites, metastatic sites, and/or a disseminated tumor cell pool, these cells have clinical significance. The circulating tumor cells offer a rare glimpse of the evolution of the tumor and its response/resistance to treatment in a real-time non-invasive manner. Although the clinical relevance of circulating tumor cells is undeniable, the routine use of these cells remains limited due to the elusive nature of the cells, which demands highly sophisticated and costly instrumentation. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of circulating tumor cells from peripheral blood of 91 consented and enrolled patients with tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Our user-friendly cost-effective circulating tumor cells detection technique has the potency to facilitate the routine use of circulating tumor cells detection even in community-based cancer centers for prognosis, before and after surgery, which will provide a unique opportunity to move cancer diagnostics forward. ABSTRACT: The source of circulating tumor cells (CTC) in the peripheral blood of patients with solid tumors are from primary cancer, metastatic sites, and a disseminated tumor cell pool. As 90% of cancer-related deaths are caused by metastatic progression and/or resistance-associated treatment failure, the above fact justifies the undeniable predictive and prognostic value of identifying CTC in the bloodstream at stages of the disease progression and resistance to treatment. Yet enumeration of CTC remains far from a standard routine procedure either for post-surgery follow-ups or ongoing adjuvant therapy. The most compelling explanation for this paradox is the absence of a convenient, laboratory-friendly, and cost-effective method to determine CTC. We presented a specific and sensitive laboratory-friendly parallel double-detection format method for the simultaneous isolation and identification of CTC from peripheral blood of 91 consented and enrolled patients with various malignant solid tumors of the lung, endometrium, ovary, esophagus, prostate, and liver. Using a pressure-guided method, we used the size-based isolation to capture CTC on a commercially available microfilter. CTC identification was carried out by two expression marker-based independent staining methods, double-immunocytochemistry parallel to standard triple-immunofluorescence. The choice of markers included specific markers for epithelial cells, EpCAM and CK8,18,19, and exclusion markers for WBC, CD45. We tested the method’s specificity based on the validation of the staining method, which included positive and negative spiked samples, blood from the healthy age-matched donor, healthy age-matched leucopaks, and blood from metastatic patients. Our user-friendly cost-effective CTC detection technique may facilitate the regular use of CTC detection even in community-based cancer centers for prognosis, before and after surgery.