ArticleBio-protocol2026
Generation and Characterization of Adaptive Anoikis-Resistant Cells Using Cyclic Attachment-Detachment Culture of Cancer Cells.
Article in Bio-protocol, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Anoikis resistance, or the ability of cancer cells to evade cell death triggered by immediate detachment from the extracellular matrix, is a critical established hallmark of metastatic cancer. While suspension culture models have been used to study anoikis, most focus on defined single time points or prolonged suspension that may not recapitulate the effects of repeated stress that tumor cells experience during metastatic dissemination. Here, we describe a detailed protocol for generating anoikis-resistant (AnR) cancer cells that have adapted to such stress through exposure to repeated cycles of suspension stress on poly-HEMA-coated plates, followed by recovery under standard attached conditions. The protocol includes methods for determining baseline anoikis sensitivity, generating AnR cells over 7-9 attachment-detachment cycles, assessing the stability and reversion of the anoikis-resistant phenotype, and characterizing AnR cells using Live/Dead staining of spheroids, flow cytometry-based apoptosis assays, and immunofluorescence for proliferation markers. This approach produces a non-genetic, reversible anoikis-resistant state that models the adaptive transcriptional reprogramming underlying metastatic progression, providing a reproducible and physiologically relevant in vitro system for studying anoikis resistance mechanisms and evaluating therapeutic strategies for prevention and reversal of such adaptations. Key features • Generates adapted anoikis-resistant cancer cells through cyclic attachment-detachment culture on poly-HEMA-coated plates over 7-9 passages. • Produces a non-genetic and reversible resistant state that models adaptive transcriptional reprogramming during metastatic dissemination. • Includes methods for assessing anoikis resistance stability, reversion kinetics, and cryopreservation of cells at defined passages. • Provides three complementary characterization assays: Live/Dead spheroid imaging, Annexin V/PI flow cytometry, and Ki67 immunofluorescence.
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