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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.

Miao Huang,1,* Yuanyuan Ma,1,* Chao Lv,1 Shaolei Li,1 Fangliang Lu,1 Shanyuan Zhang,1 Daisy Dandan Wang,2 Peter Ping Lin,2 Yue Yang1 1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Thoracic Surgery II, Peking University Cancer Hospital & Institute, Beijing, People’s Republic of China; 2Department of Oncology, Cytelligen, San Diego, CA, USA*These authors contributed equally to this workCorrespondence: Yue YangKey Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Thoracic Surgery II, Peking University Cancer Hospital & Institute, 52 Fucheng Road, Haidian District, Beijing, 100142, People’s Republic of ChinaTel +86 10 8819 6568Email yangyue236@outlook.comPurpose: This study aimed to explore the potential application of circulating tumor cells (CTCs) in predicting the therapeutic effect of neoadjuvant chemotherapy (NAC) in non-small-cell lung cancer (NSCLC).Methods: Using integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization, the serial CTCs of patients with NSCLC were detected in 7.5 mL of blood at baseline and after two cycles of cisplatin-based NAC, and all aneuploidies of chromosome 8 were examined in the enriched CTCs. Tumor responses were evaluated radiologically with serial chest computed tomography (CT) using the response evaluation criteria in solid tumors and microscopically using the tumor cell necrosis rate (TCNR) of the resected specimen after NAC.Results: After two cycles of cisplatin-based NAC, 89% (8/9) of the patients with radiological partial response to NAC had reduced CTC numbers, while 73% (8/11) of the patients with stable disease exhibited increased CTC numbers (P = 0.0098). On pathological examination, 90% (9/10) of patients with a TCNR lower than 30% had > 1 CTC post-NAC, while 80% (4/5) of patients with a TCNR higher than 30% had ≤ 1 CTC post-NAC (P = 0.017). In aneuploidy analysis, the positive rate (CTC > 0) of triploid CTCs was found to have increased after NAC, in contrast with the tetraploid and multiploid CTCs. Furthermore, tetraploid and multiploid CTCs were found to be significantly downregulated in the patients with partial response to NAC.Conclusion: The correlations of aneuploid CTCs with both radiological and pathological responses in patients with NSCLC who received NAC were summarized, and the findings indicate that enumerating and karyotyping aneuploid CTCs can serve as a surrogate marker for disease monitoring in NSCLC.Keywords: non-small-cell lung cancer, circulating tumor cell, neoadjuvant chemotherapy, therapeutic response

ObjectiveThe size distribution of circulating aneuploid cells (CACs) and its clinical significance were investigated in resectable non-small cell lung cancer (NSCLC).Patients and MethodsA total of 50 patients with resectable NSCLC were enrolled in this study. Blood samples (50 pre-surgery and 35 post-surgery) were collected and used for the detection of CAC chromosome 8 heteroploidy through the subtraction enrichment and immunostaining fluorescence in situ hybridization (SE-iFISH) method.ResultsLess than 20% small cell size and more than 80% large cell size CACs were detected. Karyotypes, including triploid, tetraploid, and multiploid, had varying distributions. The triploid subtype accounted for the majority of small cell size CACs, whereas the multiploid subtype accounted for the majority of large cell size CACs. We found that total small cell size and triploid small cell size CACs, but not large cell size CACs, derived from pre-surgery samples, were associated with shorter disease-free survival. Moreover, total small cell size and triploid small cell size CACs were associated with higher TNM stage and recurrence. Nevertheless, the variation between pre- and post-surgery CACs was not related to survival among patients with resectable NSCLC.ConclusionsPre-surgery small cell size CACs, especially the triploid subtype, could be regarded as a potential prognostic biomarker for patients with resectable NSCLC.