Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

Abstract

Background Graft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products. Methods Neuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture. Results PDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft. Conclusion These results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Label-Free Separation of Circulating Tumor Cells and Clusters by Alternating Frequency Acoustic Field in a Microfluidic Chip

“…Even when the flow rate was increased to 900 μL/h, the separation efficiency was around 84% and the PBMCs contamination rate remained unchanged ( Figure 4 ). Compared with other separation methods based on acoustofluidic, the usual flow rate was 120–6000 μL/h, the separation efficiency of cancer cell lines was 71–98%, and the PBMCs contamination rate was 0.2–10% [ 25 , 37 , 38 , 39 , 40 , 41 , 42 ]. It is worth noting that the higher flow rates reported in some studies were due to the larger cross-section of the microchannel (such as five times larger than our [ 39 ]) and higher intensity of acoustic field, which will be improved in our next optimization.…”

Pro-metastatic and mesenchymal gene expression signatures characterize circulating tumor cells of neuroblastoma patients with bone marrow metastases and relapse

“…Suboptimal CTC enrichment may reflect limitations with current early-generation microfluidic technology, particularly for low volume blood samples and BMAs. In future, advanced single-cell capture and rare cell enrichment methods may facilitate the study of CTC biology in vitro and in CTC-derived xenograft models ( 6 , 25 , 66 – 68 ).…”

Circulating tumor cells in neuroblastoma: Current status and future perspectives

“…Antigen‐independent methods are based on differences in physical properties between CTCs and non‐malignant blood cells such as cell size, density, electric charges, and deformability. One study has developed acoustophoresis, a label‐free microfluidic technology utilizing ultrasound waves in microchannels, to separate NB tumor cells from peripheral blood stem cells (PBSC) based on different cell size distribution between malignant and normal cells 36 . However, this method was limited by low throughput with 10 5 cells/min and limited purity out of heterogeneity in NB tumor cell size.…”