Abstract
Background Tumor-associated macrophages (TAMs) are known to support tumor growth by suppressing the activity of tumor infiltrating T cells. Consequently, the number of TAMs has been correlated with a poor prognosis of cancer. However, the molecular reason why TAMs acquire an immunosuppressive phenotype is still not completely understood. During solid tumor growth, the extracellular matrix is degraded and substituted with a tumor specific high-density collagen-rich extracellular matrix. The collagen density of the tumor extracellular matrix has been associated with a poor prognosis of several cancers, but the reason for this is still unknown. Here, we have investigated if the pro-tumorigenic effects of collagen density could involve the modulation of TAM functions.
Methods In this study, the macrophage cell line RAW 264.7 was cultured in 3D collagen matrices of low- and high collagen densities mimicking healthy and tumor tissue, respectively. Using this model, the effects of collagen density on macrophage phenotype and function were investigated by confocal microscopy, flow cytometry, RNA sequencing, qRT-PCR, and ELISA analysis. To investigate the effect of collagen density on the immune modulatory activity of macrophages, co-culture assays with primary T cells to assess T cell chemotaxis and T cell suppression were conducted.
Results Collagen density did not affect the proliferation, viability or morphology of macrophages. However, whole-transcriptome analysis revealed that the expression of several immune regulatory genes and genes encoding chemokines were differentially regulated between cells grown in low- and high-density collagen. Key findings were confirmed using qRT-PCR analysis and ELISA. Strikingly, the gene regulations had clear functional consequences. Macrophages grown in high-density collagen were less efficient at attracting cytotoxic T cells and at the same time capable of inhibiting T cells proliferation to a greater extent than macrophages grown in low density collagen.
Conclusion Our study shows that increased collagen density creates a more immunosuppressive phenotype of macrophages. This could be one of the mechanisms linking increased collagen density to poor patient prognosis.