Understanding Collagen Organization in Breast Tumors to Predict and Prevent Metastasis

Introduction: Collagen I fibers that are visible with second harmonic generation (SHG) are associated with efficient tumor cell locomotion, and preferential locomotion along these fibers correlates with a more aggressively metastatic phenotype. Through mechanisms that are not yet well understood, macrophages are able to manipulate the structure of collagen fibers in the developing murine breast with SHG as a readout of collagen fiber structure, without affecting the total amount of collagen present as quantified by immunofluorescence (IF). Tumor-associated macrophage (TAM) infiltration in breast tumors correlates with poor prognosis and increased likelihood of metastasis in the clinic, making TAMs an interesting and accessible therapeutic target. Furthermore, TAMs are known to produce tumor necrosis factor alpha (TNF-α), which has differential effects on breast cancer cells and stromal fibroblasts, and treatments known to affect macrophage cytokine production have likewise been shown to affect tumor SHG. Taken together, these facts suggest TAMs and stromal expression of TNF-α may play a role in collagen organization in the tumor. Methods: We compared SHG and IF signals from tumors grown in mice with and without TNF-α, in the presence or absence of TAMs. Results were compared with t-tests and ANOVA as appropriate. Results: Using liposome-encapsulated clodronate to specifically deplete TAMs, we show for the first time that modulation of TAM presence alters tumor collagen fibrillar structure on a molecular level as quantified by SHG and IF. This alteration in structure correlates with a decrease in lung metastases. Furthermore, we show that abrogation of TNF-α expression by tumor stromal cells also alters fibrillar structure, and that subsequent modulation of TAM presence has no effect on these signals. In each case, metastatic burden again correlates with optical readouts of collagen structure. Conclusions: Our results implicate TAMs and stromal TNF-α as a regulator of collagen structure in the metastatic breast tumor, and suggest that this structure may in turn play a role in tumor metastasis. These findings represent a novel mechanistic role by which TAMs exert tumorandmetastasis-promoting effects in vivo to complement and amplify their alreadycharacterized immunological functions.