Influence of EDA fibronectin on macrophages and tumor growth
Final Report Abstract
Fibronectin is an extracellular matrix protein produced by and released from almost all mammalian cells. It is also detected in the circulation, as it is released from hepatocytes in the liver. In patients with cancer, its presence is associated with poor prognosis (earlier death). In mice, a decrease in fibronectin, irrespective of the reason, leads to smaller tumors due to decreased angiogenesis (new blood vessel formation) and suppressed tumor cell proliferation. We had detected a difference in the degree of cancer suppression depending on the isoform of fibronectin involved. Loss of total fibronectin results in an increase in macrophage infiltration in the tumor presumably leading to a more pronounced immune rejection. Furthermore, loss of the isoform containing the extra domain called extra domain A (EDA) changed myeloid cell differentiation in the bone marrow and ultimately their behavior leading to an enhanced immune response and less cancer growth. The aims of the application were therefore to characterize the changes in immune cells that are attributed to loss of either the EDA or a second extra domain called EDB or both domains, determine whether indeed these immune cells are differently affected depending on the isoform they are exposed to and understand the mechanism involved. Another aim was to try to modulate the amount of fibronectin and its isoforms in the tumor pharmacologically in order to prevent tumor progression. Depletion of CD11b+ cells (using endosomes containing clodronate) in mice injected with tumor cells unable to produce fibronectin led to growth similar to that using cells able to produce large amounts of fibronectin. This suggested that CD11b + cells mediate the -early- suppression of growth in the absence of fibronectin. Using CRISPR/Cas9, we deleted either EDA, EDB or both domains from tumor cell fibronectin. A population expressing Ly6G+ was larger in the absence of both domains. We therefore first depleted Ly6G+ cells and found that only in the absence of both domains the markedly suppressed cancer growth increased back to that of control cells. This established that in the absence of both extra domains, Ly6G+ cells result in an immune response that inhibits cancer growth. Furthermore, a subtype of M2-macrophages was increased in the presence of EDB. Proteome analysis of sorted Ly6G+ cells and macrophages revealed a change in several molecules. Three were chosen as potential mediators of Ly6G-mediated suppression of tumor growth in the absence of both domains and three as mediators of the effect of EDB on the macrophages. This work is in its final stages before submission for publication. Pharmacologic modulation of fibronectin as well as collagen provided further insight. Preventing accumulation of fibronectin suppressed tumor progression in two models. Even though it did not shrink the tumors, the effects were more pronounced in smaller tumors that barely grew after the start of treatment. The slowing down of progression was measurable in large tumors too, however. Manipulation of collagen I on the other hand showed no effect. The mechanism for suppression was at least in part mediated by changes in expression and phosphorylation of transcription factors. In summary, the data generated through the funding confirm that pharmacologic modulation of the extracellular matrix protein fibronectin is able to slow cancer progression, particularly in small tumors, but also suggest that a better understanding of how fibronectin isoforms modulate the immune response is a worthwhile endeavor. Several leads in the modulation of the immune response against cancer seem promising.
Publications
- "A subpopulation of stromal cells controls cancer cell homing to the bone marrow". Cancer Res. 2018 Jan 1;78(1):129-142
Rossnagl S, Ghura H, Groth C, Altrock E, Jakob F, Schott S, Wimberger P, Link T, Kuhlmann JD, Stenzl A, Hennenlotter J, Todenhöfer T, Rojewski M, Bieback K, Nakchbandi IA
(See online at https://doi.org/10.1158/0008-5472.can-16-3507) - "Fibronectin and Its Receptors in Hematopoiesis". Cells. 2020 Dec 18;9(12):2717
Wirth F, Lubosch A, Hamelmann S, Nakchbandi IA
(See online at https://doi.org/10.3390/cells9122717) - "The Rho GTPase RAC1 in Osteoblasts Controls Their Function". Int J Mol Sci. 2020 Jan 8;21(2):385
Huck K, Sens C, Wuerfel C, Zoeller C, Nakchbandi IA
(See online at https://doi.org/10.3390/ijms21020385) - "Cdc42 in osterixexpressing cells alters osteoblast behavior and myeloid lineage commitment". Bone. 2021 Dec;153:116150
Wirth F, Huck K, Lubosch A, Zoeller C, Porubsky S, Nakchbandi IA
(See online at https://doi.org/10.1016/j.bone.2021.116150) - "Inhibition of fibronectin accumulation suppresses tumor growth". Neoplasia. 2021 September;23(9):837-850
Ghura H, Keimer M, von Au A, Hackl N, Klemis V, Nakchbandi IA
(See online at https://doi.org/10.1016/j.neo.2021.06.012)
