Tumour cell‐secreted factors skew infiltrating immune cells towards a tumour‐supporting phenotype, expressing pro‐tumourigenic mediators. However, the influence of lipocalin‐2 (Lcn2) on the metastatic cascade in the tumour micro‐environment is still not clearly defined. Here, we explored the role of stroma‐derived, especially macrophage‐released, Lcn2 in breast cancer progression. Knockdown studies and neutralizing antibody approaches showed that Lcn2 contributes to the early events of metastasis in vitro. The release of Lcn2 from macrophages induced an epithelial–mesenchymal transition programme in MCF‐7 breast cancer cells and enhanced local migration as well as invasion into the extracellular matrix, using a three‐dimensioanl (3D) spheroid model. Moreover, a global Lcn2 deficiency attenuated breast cancer metastasis in both the MMTV–PyMT breast cancer model and a xenograft model inoculating MCF‐7 cells pretreated with supernatants from wild‐type and Lcn2‐knockdown macrophages. To dissect the role of stroma‐derived Lcn2, we employed an orthotopic mammary tumour mouse model. Implantation of wild‐type PyMT tumour cells into Lcn2‐deficient mice left primary mammary tumour formation unaltered, but specifically reduced tumour cell dissemination into the lung. We conclude that stroma‐secreted Lcn2 promotes metastasis in vitro and in vivo, thereby contributing to tumour progression. Our study highlights the tumourigenic potential of stroma‐released Lcn2 and suggests Lcn2 as a putative therapeutic target. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

While the importance of iron for tumor development is widely appreciated, the exact sources of tumor-supporting iron largely remain elusive. The possibility that iron might be provided by stromal cells in the tumor microenvironment was not taken into account so far. In the present study, we show that tumor-associated macrophages (TAM) acquire an iron-release phenotype upon their interaction with tumor cells, thereby increasing the availability of iron in the tumor microenvironment. Mechanistically, TAM expressed elevated levels of the high-affinity iron-binding protein lipocalin-2 (LCN-2), which appeared to be critical for the export of iron from TAM, and in turn enhanced tumor cell proliferation. Moreover, in PyMT-mouse tumors as well as in primary human breast tumors LCN-2 was predominantly expressed in the tumor stroma as compared to tumor cells. LCN-2 expression in the stroma further correlated with enhanced tumor proliferation in vivo. Our data suggest a dominant role of TAM in the tumor iron-management and identify LCN-2 as a critical iron transporter in this context. Targeting the LCN-2 iron export mechanism selectively in stromal cells might open for future iron-targeted tumor therapeutic approaches.

Kupffer cells (KCs) are tissue-resident macrophages that colonize the liver early during embryogenesis¹. Upon liver colonization, KCs rapidly acquire a tissue-specific transcriptional signature, mature alongside the developing liver and adapt to its functions^1,2,3. Throughout development and adulthood, KCs perform distinct core functions that are essential for liver and organismal homeostasis, including supporting fetal erythropoiesis, postnatal erythrocyte recycling and liver metabolism⁴. However, whether perturbations of macrophage core functions during development contribute to or cause disease at postnatal stages is poorly understood. Here, we utilize a mouse model of maternal obesity to perturb KC functions during gestation. We show that offspring exposed to maternal obesity develop fatty liver disease, driven by aberrant developmental programming of KCs that persists into adulthood. Programmed KCs promote lipid uptake by hepatocytes through apolipoprotein secretion. KC depletion in neonate mice born to obese mothers, followed by replenishment with naive monocytes, rescues fatty liver disease. Furthermore, genetic ablation of the gene encoding hypoxia-inducible factor-α (HIF1α) in macrophages during gestation prevents the metabolic programming of KCs from oxidative phosphorylation to glycolysis, thereby averting the development of fatty liver disease. These results establish developmental perturbation of KC functions as a causal factor in fatty liver disease in adulthood and position fetal-derived macrophages as critical intergenerational messengers within the concept of developmental origins of health and diseases⁵.

During the course of sepsis in critically ill patients, kidney dysfunction and damage are among the first events of a complex scenario toward multi-organ failure and patient death. Acute kidney injury triggers the release of lipocalin-2 (Lcn-2), which is involved in both renal injury and recovery. Taking into account that Lcn-2 binds and transports iron with high affinity, we aimed at clarifying if Lcn-2 fulfills different biological functions according to its iron-loading status and its cellular source during sepsis-induced kidney failure. We assessed Lcn-2 levels both in serum and in the supernatant of short-term cultured renal macrophages (MΦ) as well as renal tubular epithelial cells (TEC) isolated from either Sham-operated or cecal ligation and puncture (CLP)-treated septic mice. Total kidney iron content was analyzed by Perls’ staining, while Lcn-2-bound iron in the supernatants of short-term cultured cells was determined by atomic absorption spectroscopy. Lcn-2 protein in serum was rapidly up-regulated at 6 h after sepsis induction and subsequently increased up to 48 h. Lcn-2-levels in the supernatant of TEC peaked at 24 h and were low at 48 h with no change in its iron-loading. In contrast, in renal MΦ Lcn-2 was low at 24 h, but increased at 48 h, where it mainly appeared in its iron-bound form. Whereas TEC-secreted, iron-free Lcn-2 was associated with renal injury, increased MΦ-released iron-bound Lcn-2 was linked to renal recovery. Therefore, we hypothesized that both the cellular source of Lcn-2 as well as its iron-load crucially adds to its biological function during sepsis-induced renal injury.

Macrophages supply iron to the breast tumor microenvironment by enforced secretion of lipocalin-2 (Lcn-2)-bound iron as well as the increased expression of the iron exporter ferroportin (FPN). We aimed at identifying the contribution of each pathway in supplying iron for the growing tumor, thereby fostering tumor progression. Analyzing the expression profiles of Lcn-2 and FPN using the spontaneous polyoma-middle-T oncogene (PyMT) breast cancer model as well as mining publicly available TCGA (The Cancer Genome Atlas) and GEO Series(GSE) datasets from the Gene Expression Omnibus database (GEO), we found no association between tumor parameters and Lcn-2 or FPN. However, stromal/macrophage-expression of Lcn-2 correlated with tumor onset, lung metastases, and recurrence, whereas FPN did not. While the total iron amount in wildtype and Lcn-2^−/− PyMT tumors showed no difference, we observed that tumor-associated macrophages from Lcn-2^−/− compared to wildtype tumors stored more iron. In contrast, Lcn-2^−/− tumor cells accumulated less iron than their wildtype counterparts, translating into a low migratory and proliferative capacity of Lcn-2^−/− tumor cells in a 3D tumor spheroid model in vitro. Our data suggest a pivotal role of Lcn-2 in tumor iron-management, affecting tumor growth. This study underscores the role of iron for tumor progression and the need for a better understanding of iron-targeted therapy approaches.

Dynamical models in the form of systems of ordinary differential equations have become a standard tool in systems biology. Many parameters of such models are usually unknown and have to be inferred from experimental data. Gradient-based optimization has proven to be effective for parameter estimation. However, computing gradients becomes increasingly costly for larger models, which are required for capturing the complex interactions of multiple biochemical pathways. Adjoint sensitivity analysis has been pivotal for working with such large models, but methods tailored for steady-state data are currently not available. We propose a new adjoint method for computing gradients, which is applicable if the experimental data include steady-state measurements. The method is based on a reformulation of the backward integration problem to a system of linear algebraic equations. The evaluation of the proposed method using real-world problems shows a speedup of total simulation time by a factor of up to 4.4. Our results demonstrate that the proposed approach can achieve a substantial improvement in computation time, in particular for large-scale models, where computational efficiency is critical.