Convincing evidence supports the involvement of inflammatory components in different stages of cancer development, including cancer, cancer invasion and metastasis. However, allergen-induced airway inflammation characterized by significant infiltration of eosinophils into the site of inflammation is negatively correlated with cancer risk in epidemiological and experimental studies. Eosinophil infiltration is also frequently observed in various solid tumors in clinical practice and is often associated with a good prognosis in most studies, suggesting that eosinophils have potential antitumor effects. This has triggered a large number of studies to address the underlying mechanisms involved. Several studies have demonstrated the antitumor function of eosinophils in vivo and in vitro and highlighted various mechanisms, generalized as (i) degranulation of cytotoxic particles and enzymes to directly kill tumor cells, and (ii) generation of broad immunoactive factors to regulate the tumor environment. However, little information is available on the direct effect of eosinophils on tumor metastasis.

Eosinophil-associated inflammation in the tumor microenvironment may also lead to metastasis. Eosinophil-associated cytokines, such as interleukin-5 (il-5), are key factors affecting eosinophil differentiation and survival in allergic inflammation. Although confounding factors complicate the relationship between eosinophils and tumors, literature has shown that il-5 forms metastatic colonization of lungs through eosinophils. It has been reported that eosinophils (epx) promotes tumor metastasis in the 4T1 breast cancer model. By tissue staining, epx is also considered a potential biomarker for predicting clinical outcomes in patients with primary lung adenocarcinoma. These studies show that eosinophils and their mediators may have a much greater role in cancer metastasis than previously known. Therefore, the anti-tumor effects of eosinophils should be reconsidered until more evidence of the mechanism of action of eosinophils in cancer metastasis.

It has been reported that c-l6) is a chemokine mainly produced in monocytes or macrophages. L6 can accelerate tumor growth. L6 is homology with the human c-c chemokine mpif-l23. The high expression of mpif-l23 is related to the accelerated progression and short survival of cancer patients. L23 binds and activates c-c chemokine receptor 1        (ccr1), expressed on the surface of a variety of immune cells and tumor cells, promoting tumor metastasis. Here, the r1 signaling pathway is involved in eosinophil-related tumor metastasis. Therefore, we studied the effect of eosinophils on experimental tumor cell metastasis under the action of inflammation, and the growth of l6-dependent metastatic tumors.

Research results

1. Ovalbumin-induced airway inflammation and glucan sodium sulfate-induced colonic inflammation promote metastasis in mice

To determine the effect of inflammation accompanied by eosinophilia on tumor metastasis, we first established a melanoma lung metastasis model after the typical ovalbumin (Ova)-induced asthma model in c57bl/6 wild-type (WT) mice. We observed a significant increase in lung metastasis in airway inflammation mice compared with control mice.

An experimental model of colitis induced by dextran sodium sulfate (dss) was then established, and tumor cells were injected into the abdominal cavity. In addition, DSs-induced colon inflammation leads to an aggravated phenotype of colon metastasis.

The role of alleviating inflammation to prevent metastasis is then explored. The broad-spectrum anti-inflammatory drug dexamethasone (dex) can effectively inhibit airway inflammation to reduce lung metastasis.

To confirm eosinophilia, we performed immunohistochemical and flow cytometry analysis of lung tissue in the Ova model and colon in the DSS model. The results determined eosinophil infiltration in airway inflammation.

dss     Induced colitis is manifested as a large number of leukocyte infiltration in the colonic mucosa, including eosinophils.

Eosinophil deficiency mice attenuated lung metastasis after OVA stimulation, while they had no effect on colon metastasis after DSS                                                                                                                                                                                                                                             �

2. Eosinophilia is essential for increased bone metastasis in cd3δ-il-5.

Next, to study the long-term effects of persistent eosinophilia on in vivo metastasis, we used cd3δ-il-5 transgenic (il-5tg) mice, which produced very high levels of eosinophils in the bone marrow as shown in Figure 2a and maintained in peripheral blood. Tumor cells were injected through the tail artery to transport tumor cells to organs downstream of the common iliac artery, mainly in the lower spine and hind limbs. In contrast to the fewer spine metastasis in WT     mice, we observed more visible pigmented spinal lesions in the il-5                                                                                                                                                                             �

It was reported that tail vein injection of tumor cells rarely leads to bone metastasis. However, in addition to the greater number of spinal metastasis, the risk of metastasis in il-5   Tg mice was significantly higher. Similar results were confirmed in the tibia and femur. Histological examination of the femur showed early colonization and obvious tumor lesions in il-5  Tg mice.

Given that il-5    has been shown to support metastasis by regulating multiple host cells, including eosinophils, regulatory T        natural killer cells, macrophages and neutrophils, we next identified the link between increased bone metastasis and eosinophils. To this end, we crossed il-5    tg mice with eos-null mice to eliminate eosinophils. We then used wt,il-

BM reconstruction of 5tg, il-5tg and eos-null mice was irradiated with WT mice to exclude the contribution of BM stromal cells to bone metastasis. After injection of tumor cells, chimeric mice receiving BM from il-5    Tg and eos-null donors did not show an increase in metastasis number and tumor burden. These results confirm that eosinophilia is essential for increasing bone metastasis in il-5tg mice.

3. Eosinophils directly support tumor cell migration and metastasis formation

Given the observation of enhanced bone metastasis in early il-5tg mice, we investigated whether eosinophils can directly promote the survival and growth of tumor cells. We investigated whether eosinophils can directly promote the survival and growth of tumor cells. By conducting in vitro experiments, in the absence of eosinophils or the presence of eosinophils, we found that the clonal activity of tumor cells was not significantly changed. Considering that eosinophils may induce apoptosis and necrosis of tumor cells, we evaluated the survival of tumor cells co-cultured with eosinophils. No significant effect of eosinophils on tumor cell death was found. Similar findings showed that with or without eosinophils, there was no significant difference in the proportion of edu-positive proliferating tumor cells, and there was no change in the cell cycle of tumor cells. These results suggest that eosinophils had no substantial effect on altering tumor cell growth.

Subsequently, we conducted migration experiments in vitro. During the culture process, after direct or indirect contact with eosinophils, the migration ability of tumor cells was significantly enhanced, suggesting that eosinophils promote the migration of tumor cells in a contact-independent manner. Migration tests were used to perform BM supernatant from il-5    Tg mice and control mice. The research results showed that eosinophil-derived factors may promote tumor cell migration.

We mixed tumor cells with eosinophils and then co-injected the mixture into wt      mice, which resulted in increased lung metastasis compared to injection of tumor cells alone. In parallel experiments, eosinophils were adopted to transfer immediately after intravenous injection of tumor cells (intratracheal) which led to similar results.

Next we studied the relationship between eosinophils and metastasis in cancer patients. Compared with pleural effusions in non-cancer patients, more eosinophils were detected in malignant pleural effusions (MPs) in patients with pleural metastasis (Figure 4, a and b), and other immune cells including macrophages, lymphocytes and neutrophils were not changed.

l6 is crucial in eosinophil-induced tumor cell migration and eosinophil-dependent metastasis formation

Our goal was to determine the effective factors for eosinophils to promote tumor cell migration and metastasis formation. Cytokines and chemokines such as l8, cxcl12, cxcl14 target tumor cells directly or indirectly to promote metastasis. We used real-time quantitative polymerase chain reaction (RT-QPCR) from il-5tg mice and WT mice to be the most abundant cytokine in il-5     TG mice. The human homologous gene of l6 is at level of l23, and found that the concentration of l23 is increased compared to pleural effusions in non-cancer patients (Figure 4D). The high secretion of l6 is mainly found in eosinophils (Figure 4E). Whether l6 is involved in tumor cell migration and metastasis, we generated ccl6                                                                                                                                                 tg                                                                                                                                                                                                                                                              

L6 deletion did not affect baseline lung metastasis in vivo (Figures s10, c and d), and L6 deletion significantly reduced eosinophil-dependent lung metastasis. Compared with il-5tg mice, il-l6/mouse also had much less bone metastasis after injecting tumor cells through the tail artery. In addition, after intravenous injection of tumor cells, the increase in bone metastasis number and tumor burden were reduced in ccl6-deficient IL-5tg mice. In short, l6 is crucial in eosinophil-induced tumor cell migration and eosinophil-dependent metastasis formation in vivo.

5.r1 can reduce the formation of tumor cell migration and metastasis

l6     Receptor.l6    R1     Recruiting tumor cells to the site of metastasis. We stably knocked out b16-f10    R1 by shRNA, lentiviral non-targeting    ShrNA    R1      Defective b16-f10(r1     B16-f10)r1     Defective b16-f10           Defective b16-f10                           Defective b16-f10                                                                                                                       �

r1     Specific inhibitor bx471       The effect of inhibition on tumor cells. bx471        The migration of b16-f10 in vitro induces in b16-f10 cells. bx471    The bone metastasis in treated tumor cells is significantly reduced. l6       The inhibition of r1 in tumor cells is sufficient to alleviate the migration and metastasis of eosinophils induced by tumor cells. r1     Signalization may be a valuable therapeutic strategy to prevent eosinophil-dependent tumor metastasis.

Research Conclusion

Evidence suggests that inflammatory components contribute to the development of cancer. However, eosinophils involving several inflammatory diseases are not fully explored in cancer metastasis. We found that both airway inflammatory eosinophils and colon inflammation accompanied by eosinophil infiltration are associated with increased metastasis in mice. Eosinophil-rich cd3-il-5     TG mice are responsible for increased bone metastasis in eosinophil-rich cd3-il-5    TG mice. We also observed eosinophil-increasing eosinophil-influence in malignant pleural effusions in cancer patients with pleural metastasis. Mechanistically, l6 promotes tumor cell migration and metastasis formation. Gene knockout at il-5                                                                                                                                                          

Receptors) can reduce the migration and metastasis of tumor cells. Therefore, l6     dependent prometastatic activity, r1      inhibiting and represent a method to prevent metastatic disease. Based on the current research results and the above discussion, more attention should be paid to assessing tissue eosinophilia associated with metastasis in cancer patients. Here, we predict that direct targeting inflammation or blocking eosinophil-associated chemokine pathways can achieve clinical benefits in cancer patients. We also call for vigilance about the risk of metastasis and adverse prognosis caused by eosinophilia infusion. l6                                                                                                                                                                          

Research Advantages

Various mouse models and human samples were used for mutual verification, and the experimental design was rigorous and the workload was large.

Reference: After tail vein injection, it can be changed to tail artery injection, which is more likely to promote metastasis.

Research disadvantages

After injecting tumor cells intravenous or trachea, no changes were found in other types of immune cells in the pleural effusion, and further experiments were conducted to analyze whether there were differences in the changes of each immune cell in the metastatic tumor.
Chapter completed!