Abstract 5324: Isolation of small number of tumor cells for proliferation studies using DEPArray technology
Authors: Sergio Maximilian , Rossi Chiara , Stivani Valeria , Maffei Ilaria , Rossi Marco , Parisi Gianpiero , Medoro Gianni 
Published: June 2022

Abstract
Cancer is universally recognized as a heterogeneous disease. The different cell clones present in a tumor can display distinct molecular and phenotypic profiles, which may influence proliferation capabilities, metastatic potential and response to therapy. To better understand cancer progression and possibly design effective therapeutic strategies, it is fundamental to design cell-sorting technologies able to isolate rare, viable tumor cell clones from tissue or liquid biopsies. Fluorescence-activated cell sorting (FACS) is the most widely used technique to isolate cell populations from heterogeneous samples. However, FACS analysis presents technical limitations when applied to rare cell populations and/or samples with low cellularity and limitations associated with the low total number of target cells in the sample. DEParray™ PLUS (DA) is a highly automated image-based cell sorting technology capable of isolating pure, single and rare cells from heterogeneous samples. Here we evaluate the capabilities of the DA system to isolate a small number of viable tumor cells and assess the proliferation capacity of DA-sorted cells in comparison with FACS-sorted cells. Human Jurkat T lymphoblast cell line were cultured in RPMI medium and a double staining procedure was optimized to label cells with CellTraceFarRed (CTFR), division tracking dye and Live/Dead (LD) Fixable marker. CTFR/LD staining was performed for each experiment and, at days 0 and 7 of culture, CTFR and LD fluorescences were analyzed by means of BD CANTO II Flow cytometry. At day 0, FACS analysis discriminate dead from live cells, while at day 7 evaluate cell proliferation and viability after 7 days in culture. Comparison tests were carried out following two different recovery schemes: 1 recovery of 100 CTFR+/LD- cells pool and 10 recoveries of 10 CTFR+/LD- cells pool using DA Plus and BD FACSAria Fusion. For each experiment, the results were compared to cells seeded manually, which represent our unsorted control. When comparing the results obtained with a 100-cells pool, no significant difference in cell viability and proliferation were observed between DA-sorted and FACSAria-sorted cells after 7 days in culture. On the other hand, the 10 cells pool sorted with DA PLUS showed a significantly higher viability and proliferation rate compared to those sorted with FACSAria at day 7. Moreover, cells sorted by DA-technology showed less variation between experiments, highlighting a more robust performance when sorting a small number of cells. Overall, the results confirm the unique capability of the DA system to sort a small number of cells, which maintain a higher cell viability and proliferation capacity compared to those sorted with FACS. This opens up the possibility to isolate rare tumor cell clones for functional studies that can help to better understand tumor progression and response to therapy.
Citation Format: Maximilian Sergio, Chiara Rossi, Valeria Stivani, Ilaria Maffei, Marco Rossi, Gianpiero Parisi, Gianni Medoro. Isolation of small number of tumor cells for proliferation studies using DEPArray technology [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 5324.