A Dissociation-Based Seahorse XF Workflow for Quantifying Averaged Bioenergetics in Anchorage-Independent Cancer Cell...

Cell clustering during anchorage-independent growth (AIG) is a key hallmark of malignant progression, supporting survival under detachment during metastatic dissemination. However, bioenergetic profiling of cell clusters grown under AIG conditions remains technically challenging, in part because of steep nutrient and oxygen gradients and irregular cell cluster shapes that complicate Seahorse XF assay normalization. Here, we present a dissociation-based Seahorse XF96/XF Pro method that enables high-throughput quantification of the average bioenergetic state of anchorage-independent cancer cell clusters. In this workflow, clusters generated in ultra-low adherent plates are enzymatically and mechanically dissociated into a single-cell suspension immediately prior to Seahorse XF Mito Stress Test analysis on poly-D-lysine-coated 2D microplates. This approach ensures that all viable cells throughout each cluster contribute equally to oxygen consumption rate (OCR) measurements and reduces the need for complex post hoc normalization. We further apply this workflow to an in vitro metastasis model in which A549 lung cancer cells are studied as attached, detached (anchorageindependent), and reattached populations, mimicking primary tumor cells, circulating/clustered cells, and secondary lesions, respectively. Detached cells exhibit reduced mitochondrial membrane potential and diminished basal and maximal OCR, whereas reattached cells largely restore membrane potential and respiratory capacity to levels comparable to attached cells, demonstrating that detachment-induced mitochondrial dysfunction is reversible upon reattachment. These findings align with previous work showing that anchorage-independent and clustered cancer cells undergo reversible metabolic reprogramming, including suppression of oxidative metabolism, engagement of reductive pathways to support redox homeostasis, and altered ROS handling to facilitate metastatic colonization. Collectively, this dissociation-based Seahorse workflow provides a practical and accessible platform to interrogate metabolic adaptations to anchorage-independent growth and cell clustering and identify bioenergetic vulnerabilities of metastasis-relevant cancer cell states. Key features • Dissociation-based Seahorse XF Mito Stress Test enabling per-cell bioenergetic profiling of anchorage-independent cancer cell clusters grown in ultra-low adherent plates. • Overcomes structural heterogeneity by enzymatically dissociating clusters into single-cell suspensions immediately before analysis, ensuring each viable cell contributes equally to OCR measurements. • Compatible with adherent monolayer, anchorage-independent, and reattached cell populations, enabling direct bioenergetic comparison across conditions that model primary tumor, circulating, and metastatic colonization states. • Validated in the A549 lung adenocarcinoma cell line; includes PDL coating, in-house DMEM preparation, and BAM15 concentration optimization steps adaptable to other cancer cell lines.