利用多细胞聚集体（球体）进行体外癌症研究提供了一种生理学相关模型，可以密切反映复杂的肿瘤微环境，捕获实体瘤的特性，例如细胞相互作用和耐药性。在这项研究中，我们研究了肽聚集诱导的免疫原性反应 (PAIIR)，这是一种创新方法，采用我们专门设计的工程肽来诱导免疫原性细胞死亡 (ICD)。我们在三维乳腺癌肿瘤球体的背景下将 PAIIR 诱导的 ICD 与标准 ICD 和非 ICD 诱导化疗药物进行了对比。我们的研究结果表明，PAIIR 在刺激 ICD 方面的功效优于传统化疗药物。其标志是释放关键损伤相关分子模式 (DAMP)，增强树突状细胞 (DC) 对垂死癌细胞的吞噬清除，进而激活强大的抗肿瘤免疫反应。此外，我们观察到 PAIIR 会导致树突状细胞活化增加并增加抗肿瘤细胞因子的存在。这项研究不仅展示了肿瘤球体在高效高通量筛选中的实用性，而且还强调了 PAIIR 作为针对乳腺癌的强大免疫治疗策略的潜力，为更深入的探索和潜在的临床实施奠定了基础。

Utilizing multicellular aggregates (spheroids) for in vitro cancer research offers a physiologically relevant model that closely mirrors the intricate tumor microenvironment, capturing properties of solid tumors such as cell interactions and drug resistance. In this research, we investigated the Peptide-Aggregation Induced Immunogenic Response (PAIIR), an innovative method employing engineered peptides we designed specifically to induce immunogenic cell death (ICD). We contrasted PAIIR-induced ICD with standard ICD and non-ICD inducer chemotherapeutics within the context of three-dimensional breast cancer tumor spheroids. Our findings reveal that PAIIR outperforms traditional chemotherapeutics in its efficacy to stimulate ICD. This is marked by the release of key damage-associated molecular patterns (DAMPs), which bolster the phagocytic clearance of dying cancer cells by dendritic cells (DCs) and, in turn, activate powerful anti-tumor immune responses. Additionally, we observed that PAIIR results in elevated dendritic cell activation and increased antitumor cytokine presence. This study not only showcases the utility of tumor spheroids for efficient high-throughput screening but also emphasizes PAIIR's potential as a formidable immunotherapeutic strategy against breast cancer, setting the stage for deeper exploration and potential clinical implementation.