To understand the role of inflammation in cancer evolution, it is important to understand the nature of inflammation and how it contributes to physiological and pathological processes such as wound healing and infection. While this phenomenon has been discussed for more than 100 years, recent data have redefined the concept of inflammation as a critical component of tumor progression. Many types of cancer arise from inflammation [1–3, 11–13]. While we are particularly concerned with inflammation promoting the formation of tumors, it should be noted that inflammation, especially in the wound healing process, has many similarities as well as differences with tumor formation. First, the inflammation in the process of wound healing involves the formation of granulation tissues, and the stromal cells of the components need to be built. Likewise, it involves the process of angiogenesis. Both the formation of granulation tissues and angiogenesis are similar to the formation of tumor stroma [14], as both of them have similar existence in the cytokines network [15]. Second, wound healing is controlled and limited. However, we found that the tumor was uncontrollable, especially in cell proliferation and angiogenesis [1, 2, 16–18]. In the initial stages of inflammation, the body's normal regulatory mechanisms control the wound-healing process and tissue growth. This normal regulatory mechanism does not exist in a tumor.
When the tumor and wound are in one body, the inflammation of the wound interacts with the tumor. The interaction depends on the distance between them. If the tumor is far from the wound, the interaction is mainly effected by the inflammatory factors of the serum. Inflammation in the process of wound healing under the body's normal regulation, which may be in the form of cytokines or inflammatory factors in the serum delivered to the tumor, is observed. On the other hand, tumor cells can also transmit molecular signals to the region of the healing wound to affect the process of inflammation and wound healing. For instance, although the immune system in tumor patients after surgery is usually abnormal, the surgery wound would still heal well. Furthermore, the residual tumor tissue promotes wound repair and the healing process. To investigate the interaction between the tumor and the inflammatory process in wound healing, we established a stab wound on tumor-bearing mice, and expanded it everyday to ensure that wound healing remains in the early stage.
Melanoma is a leading cause of cancer-related deaths worldwide through the aggressive and complex ways of angiogenesis [19–22]. Melanoma cells have a strong cytokine-secreting ability and complex signal regulatory networks [23, 24]. The B16 melanoma cells came from the C57BL mouse, which has a normal immune system [25]. We used this animal model to determine the interaction between wound healing and cancer.
The first observation of our study is on the early stages of the wound. The tumor growth slowed down significantly until the wound was within the seven-day period of the model. We named this the tumor inhibition phase. At this phase, inflammatory factors played important roles in interfering with tumor cell proliferation by blood circulation. One of these factors is IFN-γ. Our data suggest that the serum and tumor had high levels of IFN-γ. IFN-γ is secreted from activated cells such as Th1 CD4+ T-helper cells into the tumor microenvironment. This enhanced antitumor immune responses and in turn induced the activation of macrophage cytotoxic activity [7, 26, 27]. IFN-γ increased susceptibility to apoptosis through Fas activators and cytotoxic chemotherapies in many cell types, including melanoma and colorectal carcinoma [28–30]. Through interactions with p53 and the inhibitor of apoptosis, XIAP, the ISG product XAF1 may allow APO2L/TRAIL to fully activate downstream caspases [31, 32]. IFN-γ can up-regulate tumor-associated antigens, carcinoembryonic antigen, and TAG72 both in vitro and in vivo [33]. IFNs can also inhibit angiogenesis by altering the stimuli from tumor cells and by directly inhibiting endothelial cells. Endothelial cells are inhibited in motility; they undergo coagulation necrosis in vitro, while the inhibition of angiogenesis occurs in vivo within 24 hours of tumor cell inoculation. Suppression of bFGF, also known as FGF2, is correlated with reduced vascularization and tumor growth [34]. The following are the reasons that accounted for our results. First is the tendency of the wound to release IFN-γ into the blood, transport it into the tumor, inhibit tumor growth, and promote tumor necrosis. The wound group was significantly affected as shown by the reduced tumor volume. The cross-section revealed a high percentage of necrosis.
Interestingly, the persistence of the wound after seven days (the earlier phase) showed a weakened influence on the tumor. The tumor volume began to increase gradually as compared to that in the control group. This was followed by the tumor size approaching or exceeding the size of that in the control group. In other words, in the first seven days after the wound secretes IFN-γ and the other factors, the tumor cells were inhibited. After seven days, no reduction in the level of IFN-γ was observed. This was confirmed when TGF-β was tested in serum or tumor. The trend was higher. As such, IFN-γ did not inhibit the tumor cells. We named this the "inhibition missing" phase.
Perhaps a series of cytokines could explain the contradiction of the inhibition missing phase. The cytokine TGF-β was detected in the tumor tissue in the wound group after day 7, and should have been released into blood circulation which would likely restore the growth of the tumor cells. To test whether TGF-β had contrary effects on IFN-γ, an in vitro assay was done. This proved that TGF-β has antagonism with IFN-γ, can resume the growth of tumor cells, migration, and invasion; it can also lead to the situation wherein IFN-γ reduces the activity of the tumor cells' MMPs. In this situation, the tumor cells restored growth and invasion, and avoided the inhibition of IFN-γ. The validation experiment in vivo also presented a similar effect on the tumor by IFN-γ injection. The level of TGF-β also increased significantly in the inhibition missing phase. Furthermore, the activities of MMP-2 and MMP-9 were also enhanced in the inhibition missing phase as compared to those in the inhibition phase.
TGF-β is an important mediator of tumor progression, which likewise regulates cell proliferation, migration, and invasion; it is an important cytokine involved in a variety of biological processes [35, 36]. We detected VEGF-a, bFGF, and other cytokines both in the serum and tumor tissue. However, only the expression of TGF-β up-regulated in the "inhibition missing phase," and was positively correlated to an increase in tumor size. The in vitro data proved that TGF-β can confront IFN-γ so that the tumor cells can restore proliferation and migration, and that it has the ability to resume invasion and the activity of the MMPs. The validation data in vivo also showed similar effect and phenotype. The IHC data also support this conclusion, as well as point out that Col IV is likewise regulated by the TGF-β/IFN-γ level.
In conclusion, the study has proven that when the wound and the tumor exist at the same time, there will be a new balance between TGF-β and IFN-γ. The wound, through the secretion of IFN-γ, interferes with the growth of the tumor cells and inhibits the tumor for a short period. Some tumor cells, through unknown mechanisms, use TGF-β against the IFN-γ effect in the restoration of tumor proliferation, invasion, and migration. As for the source of TGF-β, we speculated that the tumor cells mainly came from inflammatory factors such as IFN-γ adaptability to up-regulated expression, or were derived from the interaction between the tumor cells and the stromal cells. This needs further research to be conclusive. However, this study has proven that at least, in the interaction between tumor and inflammation by wounds, the existence of a new balance between TGF-β and IFN-γ not only contributes to the understanding of how tumor cells adapt to the inflammatory factor, but also provides a new basis to analyze the effects of the inflammatory process on tumors. This study also provides a reference to tumor surgery, especially in post-operative residual tumor assessment.