Immunotherapy has revolutionized cancer treatment, but low-immunogenicity tumors often escape immune detection. To address this challenge, Brenus Pharma developed the Stimulated Ghost Cells (SGC) platform, an allogeneic cancer vaccine technology that enhances immunogenicity by expanding antigen coverage. It overcomes also previous cancer vaccines challenges thanks to a standardized manufacturing process increasing access for patient. Here, we present STC-1010, a therapeutic colorectal cancer (CRC) vaccine, developed by exposing HCT116, HT-29, and LoVo cell lines to stress conditions —serum deprivation, irradiation, heat shock, and chemotherapy— mimicking standard CRC treatments and relapse conditions. The cells were then haptenated to boost immune response induction.

Initial validation in a 2D ex vivo model demonstrated STC-1010’s anti-tumor efficacy: Functional validation via ex vivo immune activation assays using monocyte-derived dendritic cells (mDCs) from several donors demonstrated STC-1010’s immunostimulatory capacity.

Vaccine-exposed mDCs showed increased IL-8 secretion, enhanced antigen processing and presentation, and activation of IL-6/JAK/STAT3 and phagosome pathways, confirmed by GSVA using KEGG/Hallmark databases. STC-1010 successfully primed mDCs, with haptenated epitopes detected. Primed CD8+ T cells induced higher apoptosis in HCT116, HT-29, and SW620 cells compared to controls. However, conventional 2D models inadequately replicate tumor complexity, contributing to high failure rates in clinical translation. To better mimic the in vivo tumor microenvironment, we developed 3D CRC spheroids by co-aggregating HCT116, HT29, SW620, and LoVo cells with colorectal fibroblasts (CCD-18Co). Tumor cells were fluorescently labeled to monitor viability in co-culture with stromal and immune cells. Spheroids were characterized by bright-field microscopy, fluorescence analysis, and histology. Quality control (QC) and release criteria were established, including tumor size evaluation and chemotherapy response. Additionally, the 3D CRC spheroid models were co-cultured with T-cells, and their activation was assessed by monitoring tumor killing and cytokine secretion. Furthermore, we developed a process to enable preservation of the 3D spheroids—an essential step toward generating off-the-shelf preserved spheroid panels for STC-1010 potency testing.

This innovation overcomes ethical, financial, and time limitations of in vivo models while preserving key tumor resistance mechanisms. Our results support STC-1010 as a promising immunotherapy and demonstrate the value of 3D spheroids as reliable preclinical tools for evaluating new treatments. Overall, this work enhances strategies to overcome immunotherapy resistance and accelerates translational research with scalable, physiologically relevant models.