Macrophage Contribution to Tumour Matrisome in Cutaneous Squamous Cell Carcinoma

To investigate the direct contribution of macrophages to the tumour matrisome in human cutaneous squamous cell carcinoma (CSCC), we established a three-dimensional spheroid-based coculture model incorporating CSCC cells, fibroblasts, and macrophages derived from the THP-1 cell line. This model also enabled comparison between interleukin-4-induced (M2-like) and interferon-γ-induced (M1-like) macrophages. RNA sequencing revealed differential expression of matrisome-associated genes, including the pro-tumorigenic chemokines CXCL6, CCL18, and CCL13, which were associated with M2-like macrophages, whereas CXCL9 was associated with the M1-like phenotype. NanoString analysis of CSCC tumours and normal skin confirmed increased expression of these chemokines in CSCC. The spheroid model also demonstrated substantial macrophage plasticity, particularly among initially M1-like macrophages. Mass-spectrometry-based proteomics identified galectin-9 (LGALS9) as the most prominently increased core-matrisome protein in spheroids. Immunohistochemical analysis of clinical samples confirmed significantly greater galectin-9 accumulation in CSCC metastases than in non-metastatic primary tumours. Analysis of publicly available single-cell RNA-sequencing data showed that LGALS9 expression was associated with CD86^high macrophages and further supported the macrophage origin of the identified chemokines. High LGALS9 expression correlated with reduced numbers of CD8⁺ T lymphocytes and increased expression of the galectin-9 receptor HAVCR2 (TIM-3) and T-cell exhaustion markers in CD8⁺ cells. Collectively, these findings suggest that galectin-9 may suppress CD8⁺ T-cell responses and contribute to an immunosuppressive tumour microenvironment. High galectin-9 expression may therefore serve as a marker of altered immune regulation in CSCC, particularly in metastatic disease.