Source And Role Of TSLP In Building Milieu In Carcinoma Foci In Pathogenesis Of Cervical Cancer

Cervical cancer, a common gynecological malignancy, is a serious threat to women’s health. The pathogenesis of cervical cancer is still almost not clear. In the recent years, research on tumor microenvironment have become hotspot. Angiogenesis aided by immune and tumor cells in carcinoma foci is indispensable in progress of cervical cancer.TSLP is a hematopoietic cytokine involved in various biological functions, including regulating innate and acquired immune response that also plays important roles in allergic dermatitis, respiratory diseases, parasitic infection and adjusting gut immunity. Our group has found that TSLP secreted by trophoblasts in early pregnancy promotes proliferation and invasiveness of trophoblasts in a dose-dependent manner and induces the regulatory Th2bias at maternal-fetal interface. The biological behavior of trophoblasts at fetal-maternal interface is similar to that of tumor cells, which means high capability of proliferation and invasiveness. Therefore, we propose that cervical cancer cells might secrete TSLP that is involved in regulating biological behavior of cervical cancer cells in autocrine and paracrine manner, which contributes to the pathological mechanisms, prevention and treatment of cervical cancer.On the basis of preliminary work, this present study is focused on whether cervical cancer cells regulate growth of themselves by secreting TSLP, and further investigate the regulating effect of TSLP secreted by cervical cancer cells on vascular endothelial cells and angiogenesis, and eosinophils infiltration in carcinoma foci.1. TSLP regulates viability of cervical cancer cells in an autocrine mannerThe expression of TSLP in tissues of cervicitis and cervical cancer and in cervical cancer cell line (Hela and was CaSki cell) was detected by immunohistochemistry and ELISA, respectively. TSLP receptor on the cell surface of Hela and CaSki cell was measured by flow cytometry. The regulation of TSLP on cell viability and expression of proliferation-related molecule Ki-67were detected by MTT and flow cytometry, respectively. It has been demonstrated that TSLP is higher expressed in cervical cancer tissue than in cervicitis tissue. Cervical cancer cell lines, Hela and CaSki, secrete TSLP in a time-dependent manner, and express TSLPR. The viability of Hela and CaSki cell is enhanced after treated with recombinant human TSLP (rhTSLP) for48or72hours. The rhTSLP promotes the expression of Ki-67in CaSki and Hela cells, and anti-TSLP or TSLPR neutralizing antibody down-regulates these effects. It is suggested that cervical cancer cells abnormally over-express TSLP that promotes viability of itself in an autocrine manner and facilitates proliferation by up-regulation of Ki-67expression, which contributes to development and progress of the tumor.2. Regulation effect of TSLP derived from cervical cancer cells on vascular endothelial cellsThe histocyte suspension was acquired after trypsinization of cervicitis and cervical cancer tissues. TSLPR expression on cell surface of vascular endothelium and Human Umbilical Vein Endothelial Cells (HUVECs) was detected by flow cytometry. HUVECs was treated by rhTSLP at various concentration, or seeded in culture system with supernatant of Hela or CaSki cells, meanwhile neutralizing antibody for TSLP or TSLPR in some wells. Thereafter, the proliferation, apoptosis and capability of tube formation of HUVECs were investigated by BrdU cell proliferation assay, Annexin V-FITC/PI double staining and tube formation assay, respectively. IL-6, IL-8and VEGF secreted by HUVECs and activation related molecules on HUVECs were detected by ELISA and flow cytometry, respectively. We have found that the ratio of TSLP positive vascular endothelial cells is higher in cervical cancer tissue than in cervicitis tissue (20.12%vs5.37%, respectively). HUVECs proliferation is promoted after treated with TSLP for72h, while blocking TSLP or TSLPR suppresses the effect of Hela and CaSki cell on HUVECs proliferation; rhTSLP up-regulates Ki-67expression in HUVECs, and blocking TSLP or TSLPR suppresses this effect induced by cervical cancer cells; TSLP has no regulatory effect on the apoptosis related molecules (Fas and FasL) in HUVECs; rhTSLP increases CD105expression, but do not influence CD62E expression. However, blocking TSLP or TSLPR down regulates the expression of CD105and CD62E on HUVECs co-cultured with Hela or CaSki cell; rhTSLP promotes HUVECs secreting IL-6, while blocking TSLP or TSLPR decreases Hela cell producing IL-6, IL-8and VEGF and down regulates CaSki secretes IL-6; rhTSLP promotes HUVECs angiogenesis, while neutralizing antibody for TSLP suppresses the stimulatory effect of Hela and CaSki cell on angiogenesis of HUVECs. Therefore, TSLP secreted by cervical cancer cells acts on vascular endothelial cells in a paracrine manner. On the one hand, it promotes HUVECs proliferation by up-regulating Ki-67expression and has no influence on its apoptosis. On the other hand, it activates vascular endothelial cells by up-regulating CD105and CD62E, which promotes cervical cancer cells and vascular endothelial cells secreting angiogenic factors such as IL-6, thereby facilitates vascular endothelial angiogenesis.3. Regulation of TSLP derived from cervical cancer cells on infiltration and function of eosinophilsTissues of cervicitis, LSIL, HSIL and cervical cancer were collected and eosinophils infiltration was evaluated by HE staining. The number and ratio of eosinophils in peripheral blood was collected, and TSLPR expressed on eosinophils surface was detected by flow cytometry. Chemotaxis of rhTSLP on eosinophils in peripheral blood was tested by chemotaxis assay. HL-60E was treated by rhTSLP or co-cultured with Hela or CaSki cell, then IL-8, FGF, VEGF and GM-CSF in the co-culture system were detected by ELISA. Molecules related to HL-60proliferation, apoptosis and functions were detected by flow cytometry. We have found that eosinophils infiltrated in the foci increases along with cervical disease progresses, but there’s no obvious difference in the number and ratio of eosinophils in peripheral blood between each group; eosinophils in peripheral blood expresses TSLP receptor; rhTSLP recruits eosinophils from peripheral blood in a dose-dependent manner; both CaSki and HeLa cell up-regulate HL-60expressing Ki-67, Bcl-2, FasL and Fas, and TSLP derived from cervical cancer cells down regulates the expression of Fas on HL-60. CaSki or HeLa cells promotes HL-60expressing HLA-DR, CD80and CD86, while TSLP secreted by CaSki or HeLa cells only down-regulate CD86on HL-60; moreover, the regulatory effect of TSLP secreted by CaSki or HeLa cells on INF-γ, TNF-a, IL-10and IL-5can be partly reversed by neutralizing antibody against TSLP or TSLPR; although HL-60does not secrete IL-8, it promotes CaSki or HeLa cell producing IL-8and up-regulates VEGF levels in the co-culture, which is partly suppressed by neutralizing antibody against TSLP or TSLPR. Therefore, eosinophils infiltrating in the cervical cancer foci present positive correlation to tumor progress; cervical cancer cells recruits eosinophils from nearby tissues by secreting TSLP, meanwhile facilitates progenitor cells of eosinophils differentiation and maturation in the foci by producing IL-5and suppresses apoptosis by down-regulating Fas expression, which together lead to more eosinophils infiltration in the foci. TSLP down-regulates CD80expression and production of IFN-y and TNF-a, as well as up-regulates IL-10and IL-5expression in eosinophils, which showes an unique cytokine pattern and microenvironment of immunologic tolerance in the foci. Moreover, TSLP derived from cervical cancer cells promotes cancer cells and eosinophils producing angiogenic factors.In conclusion, TSLP has multiple effects on cervical cancer progress:(1) TSLP promotes proliferation of cervical cancer cells by up-regulation of Ki-67in an autocrine manner;(2) TSLP facilitates proliferation, activation and angiogenesis of vascular endothelial cells in an paracrine manner;(3) TSLP recruits eosinophils in nearby tissues into the foci in paracrine manner and induces the progenitor cells to differentiation and maturation of eosinophils. TSLP further inhibits apoptosis of eosinophils, promotes IL-10and IL-5secretion and suppresses INF-y and TNF-a production. Moreover, TSLP promotes angiogenic factors IL-8and VEGF secretion in microenvironment by strengthening interaction between cervical cancer cells and eosinophils, which further contributes to angiogenesis in the foci. Our results may elucidate mechanism of tumor microenvironment regulating cervical cancer and provide a new thought for treating cervical cancer infiltrated with eosinophils.