Identification and Comprehensive Co-Detection of Necrotic and Viable Aneuploid Cancer Cells in Peripheral Blood

SIMPLE SUMMARY: Circulating tumor cells (CTCs) and CD31(+) circulating tumor endothelial cells (CTECs) constitute a unique pair of cellular circulating tumor biomarkers and play a crucial role in cancer metastasis and disease progression. Precise detection of live and necrotic aneuploid CTCs and CTECs in therapeutic cancer patients, though clinically highly demanded, has yet to be achieved in the field. The aim of this study is to develop a comprehensive strategy to effectively distinguish and co-detect viable and dead non-hematological cancer cells harboring aneuploid chromosomes and expressing various tumor markers. The innovative strategy developed in the present study will assist in an efficient assessment of therapy effectiveness, rapid detection of emerging treatment resistance and bringing in new insights into the comprehension of cancer cells in circulation. ABSTRACT: Aneuploid circulating tumor cells (CTCs, CD31(−)) and circulating tumor endothelial cells (CTECs, CD31(+)) exhibit an active interplay in peripheral blood, and play an essential role in tumorigenesis, neoangiogenesis, disease progression, therapy-resistant minimal residual disease (MRD), cancer metastasis and relapse. Currently, most CTC detection techniques are restricted to the indistinguishable quantification of circulating rare cells, including both necrotic and viable cells in cancer patients. Clinically imperative demands to distinguish and detect live and/or dead non-hematological aneuploid cancer cells in peripheral blood, which will assist in the rapid evaluation of therapeutic effects, real-time monitoring of treatment resistance longitudinally developed along with therapy and the effective detection of post-therapeutic MRD, have not yet been achieved. The integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH)-derived novel strategy was developed in this study, aiming to precisely identify and detect live and necrotic cancer cells (NC) enriched from carcinoma patients’ biofluids. The innovative SE-iFISH (NC) provides a meaningful and practical approach to co-detect various viable and necrotic aneuploid CTCs and CTECs. The detected circulating rare cells can be characterized and categorized into diverse subtypes based upon cell viability, morphology, multiple tumor markers’ expression, and the degree of aneuploidy relevant to both malignancy and therapeutic resistance. Each subtype of live or necrotic CTCs and CTECs possesses distinct utility in anti-cancer drug development, translational research, and clinical practice.