Autophagy-associated marker and stress-response changes in ASA- and 5-FU-treated 3D colorectal cancer spheroid models exposed to 3-MA or CQ

Abstract Autophagy-associated pathways are implicated in colorectal cancer (CRC) progression and therapy resistance. CRC is among the most frequently diagnosed malignancies worldwide and remains a major challenge in oncology due to its complex therapeutic management. We evaluated marker profiles and treatment-related responses in colonospheres derived from HCT116 and HT29 CRC cell lines exposed to aspirin (ASA), 5-fuorouracil (5-FU), or their combinations after pretreatment with 3-methyladenine (3-MA) or chloroquine (CQ). CRC cells were cultured as colonospheres and treated with the drugs, followed by assessment of colonosphere size and morphology via transmission electron microscopy (TEM), cell death-associated membrane permeabilization changes, proliferation, bulk metabolic readouts, including lactate and ATP levels, and bulk caspase-3/7 activity. Western blotting was performed to examine autophagy-related pathways and cell death-associated regulators, including BECN1, SQSTM1, LC3A/B-I, LC3A/B-II, and RIP3. The results showed treatment- and cell line-dependent changes in colonosphere size, membrane permeability, metabolic activity and autophagy-associated marker profiles under the applied experimental conditions. ATP levels showed treatment- and cell line-dependent changes, including marked reductions under selected ASA-containing conditions. The co-treatment with ASA and 5-FU combined with 3-MA and CQ differentially influenced colonospheres characteristics and autophagy-related proteins. These findings highlight the interplay between autophagy and cell death pathways in CRC colonosphere models and support further investigation of autophagy-targeting strategies under carefully defined experimental conditions.