A Macrophages-Enriched Head and Neck Tumor Spheroid Model to Study Foslip(®) Behavior in Tumor Microenvironment

PURPOSE: The tumor microenvironment (TME) is composed of various stromal components, including immune cells such as tumor-associated macrophages (TAMs), which play a crucial role in cancer initiation and progression. TAMs can exhibit either a tumor-suppressive M1 or a tumor-promoting M2 phenotype. First, we aimed to develop a 3D human heterotypic model consisting of head and neck squamous cell carcinoma (HNSCC) cells and different subtypes of macrophages to replicate the interactions between immune cells and cancer cells. We further investigated the behavior of Foslip(®), a liposomal formulation of temoporfin, using a macrophage-enriched 3D model. METHODS: Monocytes were differentiated into M1 and M2 macrophages, which represent two distinct subtypes. Following histological and molecular characterization, these macrophages were used to establish a 3D spheroid model of HNSCC enriched with either polarized macrophages or conditioned media. Flow cytometry and fluorescence microscopy were used to assess the accumulation and distribution of Foslip(®). The cytotoxic effect of Foslip(®)-mediated photodynamic therapy (PDT) was evaluated using flow cytometry. RESULTS: We developed heterotypic spheroids characterized by a mixed phenotype of evenly distributed macrophages. In this 3D co-culture model, both M1 and M2 macrophages showed significantly higher accumulation of Foslip(®) compared to the cancer cells. Although this differential accumulation did not drastically affect the overall PDT efficiency, spheroids generated with conditioned media exhibited a significant enhancement in photo-induced cell death, suggesting that the microenvironment could modulate the response to Foslip(®)-PDT. CONCLUSION: 3D models of HNSCC cells and macrophages provide valuable insights into the complex response of HNSCC cells to PDT using Foslip(®) in vitro. This model can be used to screen immunomodulatory nanomedicines targeting TAMs in solid head and neck tumors, either alone or in combination with standard therapies.