G-CSF-Mediated Metastatic Niche Development in the Omentum

The omentum is a common site of metastasis for many cancers originating from organs in the abdominal cavity, particularly epithelial ovarian, gastric, colorectal and pancreatic cancers [1]. The reasons for this apparent tropism are complex and multifactorial mechanisms that include anatomic features, cellular adhesion molecules, local microenvironmental factors, and immune system interactions in addition to simple passive cell accumulation. Successful omental metastasis involves a multistep cascade in which cancer cells must detach from the primary tumor, survive in the peritoneal cavity, migrate into the omentum, adhere there and finally invade and proliferate.

The anatomical structure and physiological properties of the omentum play a primary role in the migration and adhesion of metastatic cells. The normal circulation of peritoneal fluid in the abdominal cavity can transport suspended cancer cells to the omental surface due to gravitational forces and diaphragmatic motions. The extensive surface area of the omentum and its function in lymphatic drainage facilitate the buildup of fluid and cells in this region. In addition, specialized structures on the surface of the omentum, the so-called “milky patches”,” which contain immune cells and a rich network of blood vessels, serve as focal points for the colonization of cancer cells [2, 3]. Vascular structures such as high endothelial venules (HEVs) in these areas can facilitate the migration of immune cells and possibly also cancer cells from the bloodstream into the tissue [1].

The physical attachment of cancer cells to the surface of the omentum is a crucial step in the metastatic process and occurs through specific molecular interactions. Adhesion molecules such as CD44, which are expressed on the surface of cancer cells, make the initial contact by binding to ligands such as hyaluronan on the mesothelial cells lining the omentum [1]. Similarly, other adhesion receptors such as integrins (e.g. α5β1) enhance adhesion by binding to extracellular matrix (ECM) components (e.g. fibronectin) between or under the mesothelial cells [1]. In some cases, cancer cells can directly access the underlying ECM by disrupting the integrity of the mesothelial cell layer or triggering the detachment of mesothelial cells to initiate invasion.

The omental microenvironment is not just a passive adhesion surface, but rather an active supportive environment for metastatic cell survival, proliferation and invasion. Adipocytes, the main component of the omentum, provide lipids, an important source of energy for cancer cells, and promote tumor growth by secreting various signaling molecules such as adipokines [1]. Other stromal cells, such as omental fibroblasts and mesenchymal stem cells, also stimulate angiogenesis (formation of new blood vessels) by secreting growth factors (e.g. VEGF) and cytokines, and support the proliferation of cancer cells [1]. These stromal interactions are crucial for the rapid growth of metastatic lesions.

Immune cells in the omental microenvironment may also play a dual role in the metastatic process. In particular, tumor-associated macrophages (TAMs) are abundant in the omentum and play a key role in shaping the metastatic niche [1]. Although they may initially exhibit M1-like antitumor activity, they often transition to an M2-like, pro-tumor phenotype. M2 macrophages support metastatic growth by secreting factors that promote angiogenesis, remodeling the ECM, and facilitating immune evasion of cancer cells [1]. Thus, the immune landscape in the omentum often becomes immunosuppressive and favors the colonization and growth of metastatic cells.

An increasingly important mechanism in the metastatic process is the role of neutrophils and their ability to prepare the metastatic niche and capture cancer cells, particularly through the neutrophil extracellular traps (NETs) they form [1]. Various factors secreted by the primary tumor trigger the influx of neutrophils, among which granulocyte colony-stimulating factor (G-CSF) occupies a special place. In addition to its known primary function of increasing the production and release of neutrophils from the bone marrow [4], G-CSF can also be secreted by cancer cells and acts as a potent chemoattractant that stimulates the targeted migration of neutrophils into the omentum, particularly into the mammary spot regions [1]. This G-CSF-induced neutrophil accumulation is a crucial part of the formation of the “pre-metastatic niche” in the omentum before the metastatic cells arrive.

Neutrophils that accumulate in the omentum are activated by factors originating from the primary tumor or released locally. In particular, G-CSF together with other cytokines such as IL-8 and GRO have been shown to directly induce these neutrophils to undergo a specific process of cell death and NET release called NETosis [1]. This was confirmed by the presence of NET markers (e.g. citrullinated histone H3, extracellular DNA) in the omentum of patients with early-stage ovarian cancer and in mouse models with tumors that have not yet metastasized [1]. The findings of this study imply that tumor-derived substances, such as G-CSF, not only entice neutrophils to the area, but also instruct them to create NET structures that facilitate the spread of the disease to other parts of the body.

The molecular mechanism of NETosis is becoming clearer, especially under the influence of factors such as G-CSF. Stimulation of neutrophils by G-CSF, and similarly by IL-8 and GROs, activates intracellular signaling pathways and potentiates the so-called ‘respiratory burst’ leading to the production of large amounts of reactive oxygen species (ROS) [6, 7]. This action of G-CSF, alone or synergistically with other cytokines, facilitates the attainment of ROS levels that represent a critical threshold for NET formation. Elevated ROS levels trigger the activation of the enzyme peptidylarginine deiminase 4 (PAD4) in the cell nucleus [8]. The PAD4 enzyme converts arginines in proteins such as histone H3 into citrulline [9]. This citrullination leads to the cleavage (decondensation) of chromatin, and the resulting DNA strands, modified histones and granule proteins are shed outside the cell to form NET networks [5, 9]. The specificity of this process has been demonstrated by the fact that some chemokines, such as MCP-1, stimulate neutrophil migration but do not induce NETosis [1, 10].

The effect of NETs formed in the omentum on metastasis is particularly striking. These sticky, net-like structures act as highly effective physical traps for cancer cells circulating in the peritoneal fluid [1, 5]. The DNA backbone of NETs and the proteins attached to it mechanically trap cancer cells and anchor them to the surface of the omentum. This trapping mechanism is crucial for cancer cells to migrate from a liquid environment to a solid surface and initiate the colonization process. It has been experimentally demonstrated that cancer cells bind to these NET networks and that this interaction enhances cell adhesion [1]. This physical barrier and anchoring function makes NETs a central player in the omental metastatic cascade.

The metastasis-promoting effect of NETs is not limited to mechanical binding. Proteolytic enzymes such as neutrophil elastase and MMP, which are concentrated on NETs, can degrade and remodel the surrounding extracellular matrix [11]. This may facilitate the invasion of trapped cancer cells deeper into the omental tissue. In addition, NET components or other molecules released during NET formation may further pro-tumorize the local inflammatory environment or awaken dormant cancer cells, triggering metastatic growth [11]. The fact that elimination of NET structures (by genetic deficiency of PAD4 or by using PAD4 inhibitors/DNase) drastically reduces metastasis in omental tissue clearly demonstrates the causal and critical role of these structures in the metastatic process [1].

The diversity of mechanisms underlying omental metastasis highlights the complexity of this process and the difficulty of developing targeted therapies. Anatomical factors, cell adhesion molecules, interactions between stromal cells, immune cells such as macrophages and especially neutrophils, which are activated by factors such as G-CSF and form NETs, are important components that play a role at different stages of this process. The relative importance of these mechanisms may vary in different types of cancer. Therefore, all of these multifaceted mechanisms should be considered when developing therapeutic strategies to prevent metastasis.

In particular, the dual role of G-CSF, which is used as a standard of care in the treatment of chemotherapy-induced neutropenia, should be considered. On the one hand, it reduces the risk of infection by improving neutropenia, and on the other hand, it can increase the potential for omental metastases by activating neutrophils and triggering the formation of NETs [1]. In fact, some clinical trials have observed that prophylactic G-CSF use in ovarian cancer patients does not confer a survival benefit [1]. This situation emphasizes that G-CSF treatment should be administered with a careful risk-benefit assessment, especially in patients at risk of metastasis. Therapies that target NET formation or approaches that counteract the metastasis-promoting effects of G-CSF are important areas of investigation for future cancer treatments.