Study Of Pro-invasive Function Of Cancer-associated Fiborblasts Derived IL-22in Gastric Cancer And Its Underlying Mechanism

Objective:Gastric cancer (GC), one of the most common digestive malignancies, is the second leading cause of cancer-associated death worldwide. It has been reported that the5-year survival rate of advanced GC was only30～40%, owing to the marked propensity for invasion and metastasis of GC. Recently, accumulating body of literature has indicated that the progression of cancer is no longer being considered as an independent event which only correlates with uncontrollable growth potential of cancer cells. More importantly, it depends on the ability of cancer cells to recruit, to activate and subsequently to "take advantage of their surrounding stromal components, which also determines the fate of cancer development. Cancer-associated fibroblasts (CAFs), one of the major components of tumor microenvironment, can contribute to tumor growth, proliferation, survival, invasion and metastasis. CAFs may serve their tumor-promoting effect by producing various growth factors, cytokines, chemokines and proteinases, all of which acting on adjacent cancer cells in a paracrine manner. In addition, these secretory molecules can also mediate the process of angiogenesis, the formation of local inflammation as well as the remodeling and degradation of extracellular matrix. At the beginning of current study, our immunohistochemistry results revealed that IL-22positive stromal cells were increased at the invasive front of GC, however the origin and possible role of IL-22expression is yet to be determined. Additionally, the relationship between IL-22expression and clinicopathological features of GC patients needs to be clarified. In summary, we first examined the whole expression prolife of IL-22in GC tissues, and then performed a series of in vitro functional assay to investigate the possible roles of IL-22in cancer progression and underlying mechanism, aiming at providing potential target for GC diagnosis and therapy.Methods:(1) Thirty-six cases with primary GC, who did not receive any pre-operative anti-tumor treatment, were collected and then all the samples were subjected to immunohistochemistry for detecting the expression and localizationn of IL-22and its functional receptor IL-22R1.(2) Next, we also assessed the expression of p-STAT3, one of the intracellular signaling targets of IL-22.(3) The relationships among IL-22, IL-22R1, p-STAT3expression and clinicopathological features were also analyzed.(4) We investigated the expression of IL-22R1and JL-10R2mRNA and protein in several GC cell lines by using reverse transcription-polymerase chain reaction (RT-PCR) and western-blot.(5) Isolation and culture of primary gastric CAFs were performed in current study, and subsequently the concentration of IL-22in CAFs culture supernatant was measured by ELISA.(6) WST-1assay was preformed to investigate the effect of IL-22on cell proliferation in AGS and MKN28GC cell lines.(7) Trypan blue exclusion assay was preformed to determine the effect of IL-22on cell survival in AGS and MKN28GC cell lines.(8) The invasive ability of IL-22stimulation was detected by using Transwell matrigel assay.(9) Western-blot was performed to investigate the intracellular signaling in AGS and MKN28GC cell lines upon IL-22treatment.Results:(1) Immunohistochemistry demonstrated that immunoreactivity for IL-22was observed mainly in spindle or fibrous stromal cells at the invasive front of GC, and IL-22R1was expressed in the plasma membrane and cytoplasm of gastric cancer cells. Double immunostaining showed that IL-22was co-expressed in α-SMA positive stromal cells at the invasive front, suggesting that CAF cells may produce IL-22. Of the36GC cases, IL-22and IL-22R1immunoreactivity was positive in29(80.6%) and33(91.7%) cases, respectively.(2) The immunoreactivity of p-STAT3was strongly expressed in the nuclei of invasive gastric cancer cells in17cases, accounting for54.8%of all31available cases.(3) IL-22expression was significantly associated with the prevalence of lymphatic invasion, lymph node metastasis and high tumor stage, but other parameters-age, gender, tumor location, Lauren’s classification, and venous invasion-showed no significant relationship to IL-22expression. IL-22R1expression was also significantly associated with the prevalence of lymphatic invasion but not with other parameters. Of the29gastric cancers with IL-22-positivity,28(96.6%) were positive for IL-22expression, suggesting that IL-22R1expression was positively related to IL-22expression in the gastric cancer tissues investigated in the present study (P<0.05).(4) RT-PCR showed that the expression of IL-22receptors, including IL-22R1and IL-10R2was detected in all seven GC cell lines examined. The expression of IL-22R1and IL-10R2protein was also detected in AGS and MKN28GC cell lines.(5) The concentration of IL-22protein in culture supernatant was lower than the serum concentration from UC, whereas it was higher than the serum concentration from healthy control by using ELISA. This finding suggests that CAF cells are at least possible to produce IL-22protein.(6) WST-1results indicated that1-10ng/mL IL-22had no effect on the proliferation of AGS and MKN28GC cell lines.(7) Trypan blue exclusion assay demonstrated that1-10ng/mL IL-22had no effect on the survival of AGS and MKN28GC cell lines.(8) We then examined the invasive ability of gastric cancer cells stimulated with IL-22using a Matrigel invasion assay. Our results showed that When AGS and MKN28cells were stimulated with1-100ng/mL IL-22, the number of invasive cells was significantly increased in a dose-dependent manner. Moreover, this pro-invasive effect of IL-22can be abolished by adding IL-22neutralizing antibody.(9) Western-blot results indicated that IL-22stimulation activated the phosphorylation of STAT3and ERK in gastric cancer.Conclusion:In summary, IL-22mainly originated from the CAFs at the invasive front of GC. The high expression of IL-22was found to be associated with the clinicopathological features, including lymphatic invasion, lymph node metastasis and high tumor stage. In addition, IL-22-expressing CAFs isolated from human gastric cancer promote invasion of the cancer cells via activating STAT3and ERK signaling pathway. Taken together, targeting IL-22may be potential in the treatment of GC.