Abstract LB117: Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry
Authors: Zhang Qiang , Manai Marwa , Davis Andrew , Reduzzi Carolina , D’Amico Paolo , Jacob Saya Liz , Jiao Jianhua , Qin Weijun , Flaum Lisa , Behdad Amir , Cristofanilli Massimo , Shad Ami , Platanias Leonidas , Gradishar William 
Published: June 2022

Abstract
Introduction: CTCs play a critical role in breast cancer (BCa) metastases. Rapid identification of live CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic disease. However, CTCs are present at very low concentrations in the peripheral blood, and CTC enumeration is costly and technically challenging. We previously reported that cell-size based device utilizing a microfluidic cassette could be used to isolate live blood cells (Zhang et al. 2019 AACR). Here we describe a pilot study using novel microfluidic Parsortix workflow to identify live CTCs for patients with Stage IV BCa.
Methods: Four whole blood samples (7.5ml/each) were collected from each of 10 patients with stage IV BCa patients who had continued clinical progression of their disease on systemic therapy, under a Northwestern University IRB-approved study (NU20B03). All samples were stored at 15-30 °C in EDTA tubes. Live CTCs enrichment was performed using a cell size and deformability based Parsortix system (Angle) which utilizes a microfluidic based disposable cassette containing a step separation structure. The four samples from each patient were loaded onto the device and the separation procedure was started by using 4.5μm, 6.5μm 8μm and 10μm separation cassettes, respectively. After separation and cleaning, the CTCs captured by the Parsortix system were harvested by inverting the flow direction and then flushing from the cassette in 200μL PBS. The harvested CTCs were multi-stained directly by fluorescence labeled antibodies for Anti-CK-PE (specific for epithelial cells), DAPI (for nucleus), anti-CD45-APC (specific for leukocytes) (Menarini). Flow cytometry (FCM) was used to identify the CTCs which were classified as CK+, DAPI+ and CD45-. White blood cells from patient samples and the human breast cancer cell line MDA-MB-231 were used as negative and positive controls, respectively.
Results: A total of 500 and 200 MDA-MB-231 cells were processed as controls, which were collected completely by Parsortix and identified by FCM confirming that Parsortix system could recover the CTCs effectively. CTCs were classified based on morphology and phenotype as CK+, DAPI+ and CD45-. Live CTCs (≥1) were detected in 8 out of 10 (80%), 9 out of 10 (90%), 7 out of 10 (70%) and 5 out of 10 (50%) samples by using 4.5μm (Group 1), 6.5μm (Group 2), 8μm (Group 3) and 10μm (Group 4) separation cassettes. Maximum CTCs isolated from each group were 18, 32, 12 and 8 from Group 1, 2, 3 and 4, respectively. The corresponding average CTCs isolated from each sample were 3.5 CTCs, 5.6 CTCs, 2.5 CTCs and 1.7 CTCs from Group 1, 2, 3, and 4, respectively. The 6.5μm cassettes (Group 2) using for CTCs collection had significantly higher efficacy on collection of CTCs compared to other three groups.
Conclusions: In this pilot study, our findings demonstrated that the cell-sized dependent Parsortix system using multiple staining and FCM is an effective and specific approach for rapid recovery and identification of CTCs from Stage IV BCa patients. With further optimization, this strategy can help to accurately evaluate CTCs and offers a potential technique for diagnosis and treatmentmonitoring of patients with metastatic breast cancer.
Citation Format: Qiang Zhang, Marwa Manai, Andrew Davis, Carolina Reduzzi, Paolo D’Amico, Saya Liz Jacob, Jianhua Jiao, Weijun Qin, Lisa Flaum, Amir Behdad, Massimo Cristofanilli, Ami Shad, Leonidas Platanias, William Gradishar. Pilot study to identify live circulating tumor cells (CTCs) in metastatic breast cancer (MBC) by application of a novel microfluidic workflow system and flow cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB117.