TY  - JOUR
TI  - Generation and Characterization of Adaptive Anoikis-Resistant Cells Using Cyclic Attachment-Detachment Culture of Cancer Cells
AU  - Rajkarnikar, Resha 
AU  - Monavarian, Mehri 
AU  - Mythreye, Karthikeyan 
VL  - 16
IS  - 13
PY  - 2026
DA  - 2026/07/05
SP  - e5736
C1  - Bio-protocol 2026;16:e5736
DO  - 10.21769/BioProtoc.5736
UR  - https://doi.org/10.21769/BioProtoc.5736
AB  - 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.
KW  - Anoikis
KW  - Matrix detachment
KW  - Cell death
KW  - Cell survival
KW  - Apoptosis
KW  - Spheroid
KW  - Adaptation
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False