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

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.

Characterization and Clinical Relevance of Endometrial CAFs: Correlation between Post-Surgery Event and Resistance to Drugs

“…Two patients had aggressive TCAFs but non-aggressive NCAFs. Two out of seven aggressive CAF-bearing patients’ blood was positive for CTC, as shown using a laboratory-friendly method of double-immunocytochemistry and triple-immunofluorescence as detailed elsewhere [ 10 ]. Table 2 presents the patients’ information for the aggressive and non-aggressive CAFs.…”

Atlas of Tumor and Tumor Microenvironment Cells of Lymphovascular Space Invasion (LVSI) in High-Grade Serous Endometrial Adenocarcinoma: A Case Study

“…Although IHC can aid in the identification of LVSI in difficult or challenging cases, keeping in mind the histopathological features of our case, in line with other reports [ 13 , 14 ], we used standard H&E to aid in the diagnosis of LVSI in our case. As mentioned earlier, CTC and CAMLs were identified in our laboratory [ 15 , 16 ]. The patient’s blood was positive for both CTC and CAMLs.…”

Identification and Morphological Characterization of Features of Circulating Cancer-Associated Macrophage-like Cells (CAMLs) in Endometrial Cancers

“…CTC was determined by triple-immunofluorescence (CellSieveTM; Creatv Microtech, Potomac, MD, USA) following isolation using lithographic microfilters as presented elsewhere [ 15 ]. In short, 7.5 mL of blood was collected in CellSave collection tubes.…”

“…Parallel blood samples were spiked with AN3CA/RL-95-2/NCI-H441 tumor cells. Immunofluorescence identified CAMLs parallel to CTCs by immuno-cytochemistry (ICC), as mentioned elsewhere [ 6 , 15 ]. Details of the CellSieveTM CTC Enumeration Standard Kit (Creatv MicroTech, Inc.) are Chroma Part #49000 for DAPI, 49020 for FITC, CellSieve-PE for TRITC/PE, and CellSieve-Cy5 for Cyanine5.…”