Neutrophils are the most abundant circulating leukocytes in humans. Neutrophil infiltration into tumor tissues has long been observed but its roles have been ignored due to the presumed short life cycle and metabolic incompetence of neutrophils. Recent advances in neutrophil biology research have revealed that neutrophils have a longer life cycle with a potential to express various bioactive molecules. Clinical studies have simultaneously unraveled an increase in the neutrophil–lymphocyte ratio (NLR), a ratio of absolute neutrophil to absolute lymphocyte numbers in cancer patient peripheral blood and an association of higher NLR with more advanced or aggressive disease. As a consequence, tumor-associated neutrophils (TANs) have emerged as important players in tumor microenvironment. The elucidation of the roles of TANs, however, has been hampered by their multitude of plasticity in terms of phenotypes and functionality. Difficulties are further enhanced by the presence of a related cell population—polymorphonuclear leukocyte (PMN)-myeloid-derived suppressor cells (MDSCs)—and various dissimilar aspects of neutrophil biology between humans and mice. Here, we discuss TAN biology in various tumorigenesis processes, and particularly focus on the context-dependent functional heterogeneity of TANs.

Macrophages in lungs can be classified into two subpopulations, alveolar macrophages (AMs) and interstitial macrophages (IMs), which reside in the alveolar and interstitial spaces, respectively. Accumulating evidence indicates the involvement of IMs in lung metastasis, but the roles of AMs in lung metastasis still remain elusive. An i.v. injection of a mouse hepatocellular carcinoma (HCC) cell line, BNL, caused lung metastasis foci with infiltration of AMs and IMs. Comprehensive determination of arachidonic acid metabolite levels revealed increases in leukotrienes and PGs in lungs in this metastasis model. A 5-lipoxygenase (LOX) inhibitor but not a cyclooxygenase inhibitor reduced the numbers of metastatic foci, particularly those of a larger size. A major 5-LOX metabolite, LTB₄, augmented in vitro cell proliferation of human HCC cell lines as well as BNL cells. Moreover, in this lung metastasis course, AMs exhibited higher expression levels of the 5-LOX and LTB₄ than IMs. Consistently, 5-LOX–expressing AMs increased in the lungs of human HCC patients with lung metastasis, compared with those without lung metastasis. Furthermore, intratracheal clodronate liposome injection selectively depleted AMs but not IMs, together with reduced LTB₄ content and metastatic foci numbers in this lung metastasis process. Finally, IMs in mouse metastatic foci produced CCL2, thereby recruiting blood-borne, CCR2–expressing AMs into lungs. Thus, AMs can be recruited under the guidance of IM-derived CCL2 into metastatic lungs and can eventually contribute to the progression of lung metastasis by providing a potent arachidonic acid–derived tumor growth promoting mediator, LTB₄.

Bone metastasis is frequently complicated in patients with advanced solid cancers such as breast, prostate and lung cancers, and impairs patients’ quality of life and prognosis. At the first step of bone metastasis, cancer cells adhere to the endothelium in bone marrow and survive in a dormant state by utilizing hematopoietic niches present therein. Once a dormant stage is disturbed, cancer cells grow through the interaction with various bone marrow resident cells, particularly osteoclasts and osteoblasts. Consequently, osteoclast activation is a hallmark of bone metastasis. As a consequence, the drugs targeting osteoclast activation are frequently used to treat bone metastasis but are not effective to inhibit cancer cell growth in bone marrow. Thus, additional types of resident cells are presumed to contribute to cancer cell growth in bone metastasis sites. Cancer-associated fibroblasts (CAFs) are fibroblasts that accumulate in cancer tissues and can have diverse roles in cancer progression and metastasis. Given the presence of CAFs in bone metastasis sites, CAFs are emerging as an important cellular player in bone metastasis. Hence, in this review, we will discuss the potential roles of CAFs in tumor progression, particularly bone metastasis.

From a mouse triple‐negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of the adhesion G‐protein–coupled receptor (ADGR) family, G‐protein–coupled receptor 56 (GPR56)/adhesion G‐protein–coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1‐transduced 4T1, TS/A (mouse breast cancer cell line), or MDA‐MB‐231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1‐induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.

Patients with triple negative breast cancer (TNBC) is frequently complicated by bone metastasis, which deteriorates the life expectancy of this patient cohort. In order to develop a novel type of therapy for bone metastasis, we established 4T1.3 clone with a high capacity to metastasize to bone after orthotopic injection, from a murine TNBC cell line, 4T1.0. To elucidate the molecular mechanism underlying a high growth ability of 4T1.3 in a bone cavity, we searched for a novel candidate molecule with a focus on a transcription factor whose expression was selectively enhanced in a bone cavity. Comprehensive gene expression analysis detected enhanced Nfe2 mRNA expression in 4T1.3 grown in a bone cavity, compared with in vitro culture conditions. Moreover, Nfe2 gene transduction into 4T1.0 cells enhanced their capability to form intraosseous tumors. Moreover, Nfe2 shRNA treatment reduced tumor formation arising from intraosseous injection of 4T1.3 clone as well as another mouse TNBC-derived TS/A.3 clone with an augmented intraosseous tumor formation ability. Furthermore, NFE2 expression was associated with in vitro growth advantages of these TNBC cell lines under hypoxic condition, which mimics the bone microenvironment, as well as Wnt pathway activation. These observations suggest that NFE2 can potentially contribute to breast cancer cell survival in the bone microenvironment.

The connection between inflammation and tumorigenesis has been well established, based on a great deal of supporting evidence obtained from epidemiological, pharmacological, and genetic studies. One representative example is inflammatory bowel disease, because it is an important risk factor for the development of colon cancer. Moreover, intratumoral infiltration of inflammatory cells suggests the involvement of inflammatory responses also in other forms of sporadic as well as heritable colon cancer. Inflammatory responses and tumorigenesis activate similar sets of transcription factors such as NF-kB, Stat3, and hypoxia inducible factor and eventually enhances the expression of inflammatory cytokines including tumor necrosis factor (TNF) and chemokines. The expression of TNF and chemokines is aberrantly expressed in a mouse model of colitis-associated carcinogenesis as well as in inflammatory bowel disease and colon cancer in humans. Here, after summarizing the presumed actions of TNF and chemokines in tumor biology, we will discuss the potential roles of TNF and chemokines in chronic inflammation-associated colon cancer in mice.