Enhanced anti-tumor effects of Imatinib and immune checkpoint inhibitors on gastrointestinal stromal tumor cells (GISTs): insights from monoculture and 3D co-culture models.

Resistance to Imatinib hinders the long-term management of gastrointestinal stromal tumors (GISTs). We examined the potential of combining Imatinib with immune checkpoint inhibitors (ICIs) to augment anti-tumor efficacy and alter stress responses in GIST models. GIST cells were assessed in 2D monolayer and 3D co-culture spheroid models utilizing donor peripheral blood mononuclear cells. The treatments consisted of Imatinib administered alone and in combination with Ipilimumab and Nivolumab. MTT assays were used to evaluate cell viability, total oxidant status/reactive oxygen species (ROS), and advanced glycation end products (AGEs) assays, and mitochondrial superoxide (MitoSox) imaging to assess oxidative and glycation stress. Immunoassay and confocal microscopy were employed to measure cell proliferation (Kiel antigen 67 (Ki-67)). Western immunoblotting was performed to explore proteins involved in stress and autophagy (p62, Beclin-1). Data were expressed as mean ± SD from independent experiments (N ≥ 3); statistical significance was assessed using one-way ANOVA with Dunnett's T3 (p < 0.05). Imatinib significantly diminished GIST cell viability in both 2D and 3D models (p < 0.001), decreased intracellular ROS and AGEs, lowered Ki-67 expression, and reduced MitoSox levels. Combination therapies (Ipilimumab, Nivolumab, and combinations with Imatinib) resulted in greater reductions in 3D cell culture viability and further diminished ROS/AGEs levels compared to single agents (p < 0.001). Western immunoblotting showed that Imatinib activated autophagy (↑Beclin‑1, ↓p62). Imatinib demonstrates cytotoxic and antioxidative properties in GIST models and induces autophagy. Combining Imatinib with ICIs enhances anti-tumor effects and further reduces oxidative/glycation stress in 3D tumor-immune co-cultures. These results endorse additional preclinical and translational investigations of tyrosine kinase inhibitor immunotherapy combinations in GIST.