Depletion of circulating tumor cells using an automated device using non-hemolytic affinity based substrates

Background: While 90% cases are associated with metastatic deaths, it is imperative to monitor early stage cancer patients for the presence of systemic disease to improve overall survival (OS) and PFS. In spite of complete remission, upto 25-50 % of CRC stage II-III and early breast cancer cases are known to relapse. Further, the existence of micro-tumors using radio-imaging tools is undetected due to limit of detection. Post curative intent therapies, accounting minimal residual cellular disease (MCRD), is represented by circulating tumour cells (CTCs). CTCs are known for their extravasation and invasiveness from primary sites to distant. Further they could evade immune systems, and thus there is need to design safer extracorporeal devices for the capture and depletion of CTCs especially overexpressing PD-L1. We designed an automated device to capture and remove CTCs from whole blood. Methods: We designed an automated micro-processor operated fluidic device OncoMetastat, having cartridges for blood, reagent tubes, and a 3D-printed biocompatible spiral channel. The controller unit powers peristaltic pumps for blood circulation through the spiral channel (96 mm diameter × 6 [H] mm). It consisted an antibody and transferrin conjugated 2 mm glass beads. In addition, with 4 vibrators for micro-stirring of blood for enhanced CTC capture from 5 - 10 ml patients blood (n=54). WBC count, hemolysis and protein binding was measured. The beads were scanned for CTCs with CK18 + ve, DAP I+ ve with CD45 - ve) with an automated scanning ability and compared with OncoDiscover CTC enumeration platform approved by CDSCO India. We analyzed true positives, false negatives and sensitivity, specificity, PPV, NPV and accuracy. Results: Retrospectively, 54 pan-cancer patients' blood including breast, CRC, prostate, lung were used to capture and deplete CTCs. OncoMetastat platform demonstrated capture efficiency of > 90%, compared to OncoDiscover. Auto-scanning demonstrated 100% efficiency of CTC imaging, compared to manual imaging. Leukocyte adhesion was low with anti-EpCAM and transferrin glass beads (2 ± 1 WBCs/per sample, n = 54). WBCs enumerated showed trends specific to cancer type (mean WBC count/ml of 4.9 x 10 6 - Breast CA, 3.9 x 10 6 - Rectal CA, 3.5 x 10 6 - Prostate CA), showed decrease in WBC by 40% compared to healthy controls (mean of 6.9 x 10 6 WBCs/ml). Clinically insignificant hemolysis (<1%) and protein-binding (~1.5%) was noted. Vibration-assisted showed enhanced CTC sequestration (>90% cell capture efficiency. The sensitivity (94.4%), specificity (92.9%), PPV (94.4%), NPV (92.9%), accuracy (93.8%) was observed for CTC capture. Conclusions: We showed depletion of CTCs with specificity and efficiency. Automated device demonstrate the ability to remove CTCs from whole blood as an extracorporeal device for enhancing cancer therapy outcome.