Colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide. Most patients present with microsatellite-stable (MSS) disease that is poorly responsive to checkpoint blockade, representing a critical unmet need. We developed the Stimulated Ghost Cells (SGC) platform, an off-the-shelf immunotherapy designed to enhance tumor immunogenicity and expand antigen coverage in vivo. STC-1010 is produced by exposing CRC cell lines to clinically relevant stresses, followed by haptenation and inactivation (ghost). Previous 2D ex vivo models showed that STC-1010 activates dendritic cells, enhances antigen processing/presentation, and induces CD8⁺ T-cell–mediated tumor killing in CRC cell lines.

As conventional 2D cultures insufficiently reproduce tumor architecture and resistance mechanisms, we developed advanced 3D CRC tumor microtissue spheroid models. These models were created by co-aggregating GFP-labeled CRC cell lines with colorectal fibroblasts using InSphero technology, generating physiologically relevant structures. Spheroids demonstrated growth reflected by increased size, GFP intensity, and viability indicated by elevated ATP levels overtime. We tested STC-1010 using immune-cell–mediated cytotoxicity assays integrating DC priming, CD8⁺ T-cell activation, and multiparametric tumor-killing readouts (GFP signal reduction, DRAQ7 staining, and CD8⁺ T-cell-specific cytokine secretion). We also extended the platform to pancreatic cancer spheroids. We developed cryopreservation methods for microtissues.

Using the InSphero high-content testing platform, we measured reproducible immune-mediated anti-tumor activity while capturing donor-specific variability. Phenotypic analyses confirmed DC maturation (HLA-DR, CD86) and uptake of haptenated epitopes, supporting STC-1010’s mechanistic basis. Integrated readouts enabled robust and concordant quantification of tumor-cell death and immune effector function, strengthening assay interpretability for translational development. The pancreatic cancer spheroid model demonstrated functional responsiveness, supporting potential indication expansion. Cryopreservation studies demonstrated vitrification as the optimal method for spheroids. A workflow was refined to support future potency panels, longitudinal testing, and transferability.

Overall, these advances underscore the value of 3D spheroid systems as scalable, reproducible and physiologically relevant testing platforms that bridge in vitro and in vivo responses. Combined with early clinical progress, these data support STC-1010 as a promising immunotherapy designed to overcome immune resistance in checkpoint-refractory cancers.