Abstract LB-370: A novel ex vivo culture workflow to enrich and expand circulating tumor cells (CTCs) from patients with stage III/IV breast cancer (BCa)
Authors: Zhang Qiang , Zhang Youbin , Flaum Lisa Ellen , Helfand Brian , Gerratana Lorenzo , Gradishar William , Platanias Leonidas , Cristofanilli Massimo , Cristofanilli Massimo 
Published: August 2018

Introduction: CTCs play a critical role in BCa metastases. Molecular and genomic characterization of CTCs may help to predict BCa prognosis and identify which patients may derive treatment benefit, especially for those with metastatic or recurrent disease. However, CTCs are present at very low concentrations in the peripheral blood and their enrichment and expansion is technically challenging. Here we describe an ex vivo CTC culture workflow to expand CTCs for patients with Stage III/IV BCa.

Methods: Duplicate whole blood samples (7.5ml/each) were collected in EDTA tubes from 16 patients with stage III/IV BCa patients before or after systemic therapy and stored at 15-30 °C until processing. CTC enumeration was performed on one of the samples from each patient using the CELLSEARCH® System to confirm the presence of &gt;=5 CTCs per 7.5mL of blood. CTCs enrichment was performed on the second sample from each patient using the ParsortixTM System (ANGLE PLC), a microfluidic based technology that captures rare cells based on size and deformability. Using a proprietary Cell Separation Cassette with a critical gap size of 6.5μm, the Parsortix System captured viable CTCs from the blood inside the cassette. The CTCs were then harvested by inverting the flow direction and flushing the CTCs from the cassette into 210μL of PBS. The harvested CTCs were transferred and grown in ultralow attachment plates containing RPMI-1640 medium supplemented with EGF (20ng/ml), basic FGF (20ng/ml), B27 (10ml), and 1×Antibiotic-antimycotic. CTCs were cultured in a humid 37oC incubator with 5% CO2 and 4% O2. CTC counting was performed every week using a hemocytometer. DNA isolation of the CTC culture was performed on Day 21 using DNAzol.

Results: All 16 patients had &gt;=5 CTCs as defined by the CELLSEARCH System (based on cellular morphology and the correct phenotype of CK+, EpCAM+, DAPI+ and CD45-) at the time of the blood collection. Using the Parsortix System, highly purified CTCs (ranging in number from 300 to 17,250 cells) were isolated from the blood samples for each of the 16 patients and placed into culture (day 0). During the first week, the CTCs could be expanded to 3.5 - 5.5 fold, and then to 9.5 - 22.5 fold during the second week, to maximum amount of 112,500 within 14 days. The isolated CTCs were maintained without altered morphology at the same concentrations until Day 21. An average of 186ng and 1200ng of DNA could be isolated and purified from ~10,000 and ~110,000 cultured CTCs, respectively.

Conclusions: We identified and optimized a workflow for the recovery and culturing of CTCs from Stage III/IV BCa patients, demonstrating that cell-size dependent isolation using the Parsortix System allows for effective ex vivo culture. With further optimization, this strategy may be utilized for organoids development for drug testing and molecular analysis, representing an important tool for personalized precision therapy.

Citation Format: Qiang Zhang, Youbin Zhang, Lisa Ellen Flaum, Brian Helfand, Lorenzo Gerratana, William Gradishar, Leonidas Platanias, Massimo Cristofanilli, Massimo Cristofanilli. A novel ex vivo culture workflow to enrich and expand circulating tumor cells (CTCs) from patients with stage III/IV breast cancer (BCa) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-370.