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Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics.

ABSTRACT: Culturing and screening cells in microfluidics, particularly in three-dimensional formats, has the potential to impact diverse areas from fundamental biology to cancer precision medicine. Here, we use a platform based on anchored droplets for drug screening. The response of spheroids of Ewing sarcoma (EwS) A673 cells to simultaneous or sequential combinations of etoposide and cisplatin was evaluated. This was done by culturing spheroids of EwS cells inside 500 nL droplets then merging them with secondary droplets containing fluorescent-barcoded drugs at different concentrations. Differences in EwS spheroid growth and viability were measured by microscopy. After drug exposure such measurements enabled estimation of their IC50 values, which were in agreement with values obtained in standard multiwell plates. Then, synergistic drug combination was evaluated. Sequential combination treatment of EwS with etoposide applied 24 h before cisplatin resulted in amplified synergistic effect. As such, droplet-based microfluidics offers the modularity required for evaluation of drug combinations.

SUBMITTER: Fevre R

PROVIDER: S-EPMC10165258 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics.

Fevre Romain R Mary Gaëtan G Vertti-Quintero Nadia N Durand Aude A Tomasi Raphaël F-X RF Del Nery Elaine E Baroud Charles N CN

iScience 20230412 5

Culturing and screening cells in microfluidics, particularly in three-dimensional formats, has the potential to impact diverse areas from fundamental biology to cancer precision medicine. Here, we use a platform based on anchored droplets for drug screening. The response of spheroids of Ewing sarcoma (EwS) A673 cells to simultaneous or sequential combinations of etoposide and cisplatin was evaluated. This was done by culturing spheroids of EwS cells inside 500 nL droplets then merging them with ...[more]

PMID: 37168549

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Project description:BackgroundPediatric patients with recurrent/metastatic Ewing sarcoma (ES) have a dismal 5-year survival. Novel therapeutic approaches are desperately needed. Natural killer (NK) cell number and function are low in ES patient tumors, in large part due to the immunosuppressive tumor microenvironment (TME). Melanoma cell adhesion molecule (MCAM) is highly expressed on ES and associated with ES metastasis. NKTR-255 is a polymer-conjugated recombinant human interleukin-15 (IL-15) agonist improving NK cell activity and persistence. Magrolimab (MAG) is a CD47 blockade that reactivates the phagocytic activity of macrophages.MethodsTranscriptome profiling coupled with CIBERSORT analyses in both ES mouse xenografts and human patient tumors were performed to identify mechanisms of NK resistance in ES TME. A chimeric antigen receptor (CAR) NK cell targeting MCAM was engineered by CAR mRNA electroporation into ex vivo expanded NK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-MCAM-CAR-NK cell alone or combined with NKTR-255 against ES cells. Interferon-γ and perforin levels were measured by ELISA. The effect of MAG on macrophage phagocytosis of ES cells was evaluated by in vitro phagocytosis assays. Cell-based and patient-derived xenograft (PDX)-based xenograft mouse models of ES were used to investigate the antitumor efficacy of CAR-NK alone and combined with NKTR-255 and MAG in vivo.ResultsWe found that NK cell infiltration and activity were negatively regulated by tumor-associated macrophages (TAM) in ES TME. Expression of anti-MCAM CAR significantly and specifically enhanced NK cytotoxic activity against MCAMhigh but not MCAM-knockout ES cells in vitro, and significantly reduced lung metastasis and extended animal survival in vivo. NKTR-255 and MAG significantly enhanced in vitro CAR-NK cytotoxicity and macrophage phagocytic activity against ES cells, respectively. By combining with NKTR-255 and MAG, the anti-MCAM-CAR-NK cell significantly decreased primary tumor growth and prolonged animal survival in both cell- and PDX-based ES xenograft mouse models.ConclusionsOur preclinical studies demonstrate that immunotherapy via the innate immune system by combining tumor-targeting CAR-NK cells with an IL-15 agonist and a CD47 blockade is a promising novel therapeutic approach to targeting MCAMhigh malignant metastatic ES.

Dataset Information

Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics.

Publications

Combinatorial drug screening on 3D Ewing sarcoma spheroids using droplet-based microfluidics.

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