Abstract A017: High-throughput microfluidic enrichment of circulating tumor cells from entire diagnostic leukapheresis for comprehensive liquid biopsy
Authors: Mishra Avanish , Huang Shih-Bo , Dubash Taronish , Burr Risa , Edd Jon F. , Wittner Ben S. , Cunneely Quinn E. , Putaturo Victor R. , Deshpande Akansha , Antmen Ezgi , Gopinathan Kaustav A. , Otani Keisuke , Miyazawa Yoshiyuki , Kwak Ji Eun , Guay Sara Y. , Kelly Justin P. , Walsh John , Nieman Linda T. , Galler Isabella , Chan PuiYee , Lawrence Michael S. , Sullivan Ryan J. , Bardia Aditya , Micalizzi Douglas S. , Sequist Lecia V. , Lee Richard J. , Franses Joseph W. , Ting David T. , Brunker Patricia A. R. , Maheswaran Shyamala , Miyamoto David T. , Haber Daniel A. , Toner Mehmet 
Published: November 2024

Abstract
Circulating Tumor Cells (CTCs) in blood containing a complete set of single-cell analytes, including high-molecular-weight DNA, intact RNA, and both intracellular and cell-surface proteins serve as a promising tool in cancer diagnosis and prognosis through liquid biopsy. However, their clinical utility has been limited by their low blood concentration and inadequate enrichment technology. Diagnostic leukapheresis holds promise for isolating sufficient CTCs, but enriching ultra-rare cells from large, complex fluid volumes remains challenging. To address this, we developed the LPCTC-iChip, a high-throughput microfluidic device that uses high-flow channels and force-amplifying magnetic lenses to deplete hematopoietic cells, allowing tumor epitope-agnostic screening of massive blood volumes. Here, we present the first application of LPCTC-iChip to entire diagnostic leukapheresis products (leukopak) from seven patients with metastatic cancer, processing an average of 5.83 liters of blood per patient. Enumeration and morphology characterization of CTCs using multispectral immunofluorescence imaging identified high CTC yields - a mean of 10,057 CTCs per leukopak and showed notable intra- patient heterogeneity. Nuclear and cell diameter of CTC varies within individual patients, with 67% overlapping in size with patient-matched white blood cells. Paired single-cell DNA and RNA sequencing uncovered subclonal aneuploidy and distinct signaling pathways within CTCs with shared, clonal copy number variation (CNV) patterns. A subset of small aneuploid cells lacking epithelial markers was enriched for neuroendocrine characteristics in prostate cancer samples. The mutations found by FDA-approved genetic tests in either biopsied metastatic lesions or blood-based ctDNA sampling were reproduced by whole exome sequencing of a pool of CNV-confirmed CTCs. In addition, the sequencing revealed additional cancer-related high allele frequency variants that are not detectable with ctDNA or tissue needle biopsies. Overall, this high-throughput CTC enrichment from complete leukapheresis combined with multispectral imaging and single cell sequencing technology enables a comprehensive, cell-based liquid biopsy approach for early cancer detection and therapeutic responses monitoring.
Citation Format: Avanish Mishra, Shih-Bo Huang, Taronish Dubash, Risa Burr, Jon F. Edd, Ben S. Wittner, Quinn E. Cunneely, Victor R. Putaturo, Akansha Deshpande, Ezgi Antmen, Kaustav A. Gopinathan, Keisuke Otani, Yoshiyuki Miyazawa, Ji Eun Kwak, Sara Y. Guay, Justin P. Kelly, John Walsh, Linda T. Nieman, Isabella Galler, PuiYee Chan, Michael S. Lawrence, Ryan J. Sullivan, Aditya Bardia, Douglas S. Micalizzi, Lecia V. Sequist, Richard J. Lee, Joseph W. Franses, David T. Ting, Patricia A. R. Brunker, Shyamala Maheswaran, David T. Miyamoto, Daniel A. Haber, Mehmet Toner. High-throughput microfluidic enrichment of circulating tumor cells from entire diagnostic leukapheresis for comprehensive liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A017.