Proliferation Of Ovarian Cancer Cells Promoted By Energy Instrument Inducing Carbonized Tissue And Its Relative Mechanism

Background and Objective:Surgical treatment is the main treatment for ovarian cancer,which can clear the lesion,reduce the tumor,and make sure the diagnosis.With the improvement of surgical equipment,the use of energy instruments has become increasingly widespread.Energy instrument refers to the transformation of electrical energy or ultrasonic energy to dry and coagulate proteins so as to achieve tissue cutting and coagulation hemostasis.Energy equipment mainly includes electric knife,ultrasonic knife and so on.Although the energy instrument has good cutting and hemostasis effect and can improve the operation efficiency,the energy instrument in the practical process often leads to excessive heat in the cell,excessive tissue temperature,and even leads to different degrees of carbonization of the cutting edge and the electrocoagulation site.The components of the energy instruments inducing carbonized tissue are complex,and the main components are carbon,complex polycyclic aromatic hydrocarbons,heterocyclic amines,etc.Some studies have found that the smoke released from barbecue meat includes carbonized particles bound polycyclic aromatic hydrocarbons and other substances.These substances are deposited after entering the human respiratory tract,resulting in increased risk of cancer.Energy instruments are used in the surgery treatment of ovarian cancer.There are carbonized tissue particles in the abdominal cavity of patients.These complex carbonized tissue particles continue to exist in the abdominal cavity,and whether they have an impact on the body is worth studying.Previous studies on tissue damage caused by energy instruments mainly focus on comparing the effects and damage of different energy instruments.Even about the impact of tissue carbonization of energy instruments on the body,it is only limited to the impact of smoke generated by tissue burning of energy instruments on surgeons.Therefore,the purpose of this study is to investigate whether energy instruments inducing carbonized tissue can promote the proliferation of ovarian cancer cells and its possible mechanism.Materials and Methods:1.Six to eight weeks old female C57BL/6 mice were used to establish two kind of animal models of carbonized tissue(four treatment groups).(1)In the energy instrument operation model,the mice were subjected to abdominal operation after anesthesia.During the operation,the energy instrument was used to electrocauce the inner surface of the peritoneum to form carbonized tissue wounds with an area of about 0.5 cm~2 or 1.0 cm~2,which were respectively the Energy instrument(small area)group and the Energy instrument(large area)group.(2)The mice were intraperitoneally injected with 0.25 mg or 0.50 mg carbon particles,which were the Low dose carbon particles group and High dose carbon particles group.The non-energy instrument operation model was established as the same type control group.After anesthesia,the mice were subjected to abdominal operation,and the gauze was used to friction the peritoneal surface to form mechanical wounds during the operation.Mice with intraperitoneal injection of normal saline were the blank control group.ID8 cells(homologous mouse ovarian cancer cells)were immediately inoculated in the abdominal cavity of mice after treatment in the above groups(6 groups,7 mice in each group).Tumor tissues in the abdominal cavity of mice were weighed 42 days after tumor cells were injected.2.Detection of peritoneal tumor-associated microenvironment in mice of the NS group,Tumor group and Carbonized microparticle tumor group by flow cytometry.The peritoneal lavage fluid of mice in each group was collected 48 h after treatment to detect type 2 macrophages,monocytes,neutrophils,regulatory T cells,cytokines IFN-γsecreted by T cells,dendritic cells,Myeloid derived suppressor cells,activated T cells and CD8+T regulatory cells.3.The polarization of macrophages in related tumor microenvironment was studied as follows.(1)The peritoneal lavage fluid was collected 4 and 8 days after intraperitoneal injection of carbonized particles to detect M2 by flow cytometry,and the polarization of peritoneal macrophages in mice was studied at different time points stimulated by carbonized particles.(2)We use RT-PCR to test the expression of TGF-βm RNA and VEGF m RNA in the wound tissues of above groups of mice,four days after different kinds of surgery.(3)We use flow cytometry to test the proportion of M2 in abdominal cavity of mice in each group in the method 1.The expression of CD31 factor in tumor was detected by immunohistochemistry.4.In order to eliminate the immune response in the body,mouse peritoneal macrophages were cultured in vitro for 24 hours and 48 hours,and stimulated by different concentrations of carbon particles(0.05mg/ml,0.1mg/ml).The cultured cells were collected for FCM to detect M2,and the cytokines TGF-βand VEGF secreted by M2.Results:1.Two kind of animal models of energy instrument inducing carbonized tissue can be established stably.After 42 days of operation and inoculation with ID8,the tumor weight in the abdominal cavity of mice in the NS,Surgery,Energy instrument(small area),Energy instrument(large area)group were 0.46 g±0.12 g,0.44 g±0.14 g,0.63 g±0.14 g and 0.68 g±0.15 g.The weight of tumors in the NS group,the Low dose carbon particles group and the High dose carbon particles group were 0.43 g±0.14 g,0.52 g±0.16 g and 0.69 g±0.19 g.The proliferation of abdominal tumor in carbonized tissue animal model was significantly higher than that in the NS group and the Surgery group,and the higher the degree of carbonization,the more tumor tissue.This change is in a dose-dependent manner.2.The intra-abdominal tumor microenvironment was detected by flow cytometry 48 hours after establishment of mouse model.The percentage of neutrophils in abdominal cavity of mice in energy instrument group was higher than other groups(P<0.01).The percentage of peritoneal mononuclear cells in mice with carbonized tumor increased compared with other groups(P<0.05).The proportion of dendritic cells was higher than other groups(P<0.01).The proportion of regulatory T cells and IFN-γsecreted by T cells in the Carbon particles tumor group and Tumor group was higher than that in the NS group(P<0.01).Myeloid derived suppressor cell,activated T cells and new T regulatory cells were not significantly different in each group.The most significant difference among the groups in the related tumor microenvironment is type 2 macrophages,The relative proportions of peritoneal M2 in the NS group,the Carbon particles group,the Tumor group and the Carbon particles tumor group were 0.2600%±0.095%、1.648%±1.762%、58.360%±12.100%和77.320%±4.918%.The proportion of M2 in the Carbon particles tumor group was higher than that in the Tumor group(P<0.001),and significantly higher than the Carbon particles group and the NS group(P<0.0001).3.The research results on the polarization of macrophages are as follows:(1)After intraperitoneal injection of carbon particles,the expression of M2 in the abdominal cavity was detected at the 4th day and the 8th day.At the 4th day,the M2in the NS and the Carbon particles were 25.45%±7.04%and 43.68%±13.84%.The M2 in the Carbon particles group were more than those in the NS group(P<0.05).The M2 in the NS group and the Carbon particles group were 24.03%±5.91%and 24.91%±4.27%,and the results were not different.It shows that macrophage polarization to M2 occurs within 8 days in the presence of only carbon particles.(2)C57BL/6 mice underwent surgery or injection of carbon particles,and ID8 were inoculated for 4 days.The expression of TGF-βm RNA and VEGF m RNA in abdominal tissue or surgical wound tissue was detected.The relative expression of TGF-βm RNA in NS group,Tumor group,Surgery group,Energy instrument group and Carbon particles group were 0.00432±0.00049,0.02304±0.00347,0.07890±0.06852,0.29450±0.11230 and 0.01974±0.01118.The tissue of the Energy instrument group’s TGF-βm RNA relative expression was significantly higher than that in other groups(P<0.0001).The VEGF m RNA relative expression of the five groups were 0.02297±0.00729,0.07176±0.01645,0.15300±0.10690,0.26000±0.10400 and 0.09080±0.02853.The tissue of the Energy instrument group’s VEGF m RNA were significantly higher than those in the Carbon particles group(P<0.01),higher than those in the Tumor group(P<0.01),and higher than those in the NS group(P<0.001).(3)After the mice were treated with energy instrument surgery,non-energy instrument surgery and inoculated with ID8 cells for 42 days,M2 were detected ascites of mice in each group.The relative proportions of M2 in the NS group,Surgery group,Energy instrument(small area)group and Energy instrument(large area)group were 16.85±7.28%、29.36%±5.81%、32.40%±3.45%和41.49%±4.56%.In the Energy instrument(large area)group,the polarization of peritoneal macrophages to M2 was the most obvious,which was significantly different from that in the other three groups.(4)After 42 days of intraperitoneal injection of carbon particles and ID8 in C57BL/6 mice,M2 were detected in the cancerous ascites of mice in each group.The relative proportions of M2 in the NS group,Low dose carbon particles group and High dose carbon particles group were39.79%±1.97%,43.30%±4.79%and 53.72%±8.07%.The proportion of M2in the High dose carbon particles group was higher than that in the Low dose carbon particles group(P<0.05),and was significantly higher than that in the NS group(P<0.01).The tumor nodules in the abdominal cavity of mice in each group were taken for immunohistochemical staining of frozen sections.Each group’s CD31 was positive.The High dose carbon particles group’s CD31 expression was significantly stronger than that in the NS group and the Low dose carbon particles group.4.Vitro experimental results were as follow.M2 were detected by FCM after primary peritoneal macrophages of C57BL/6 mice were stimulated by carbon particles in vitro.The proportion of M2 in NS group and High dose carbon particles group were 7.015%±1.845%and 34.28%±1.93%at 24 h.The proportion of M2in NS group was 12.69%±3.80%,in Low dose carbon particles group was 23.47%±6.04%,and in High dose carbon particles group was 51.38%±1.61%at 48 h.The transformation of primary macrophages in the High dose carbon particles group to M2 at 48 h in vitro was higher than that at 24 h(P<0.05).We used carbon particles to stimulate primary peritoneal macrophages of C57BL/6 mice in vitro.The expression of TGF-βand VEGF secreted by M2 were detected by FCM.The expression of TGF-βin NS group,Low dose carbon particles group and High dose carbon particles group were 8.82%±3.14%,15.63%±3.32%and 20.53%±0.09%.The expression of TGF-βin High dose carbon particles group was higher than that in NS group(P<0.05).Three groups of VEGF were:3.96%±1.07%,12.76%±0.15%and 16.29%±2.41%,VEGF in the High dose carbon particles group was significantly higher than the NS group(P<0.01),The NS group was lower than the Low dose carbon particles group(P<0.05).It shows that carbon particles can stimulate macrophages in vitro and make macrophages polarized to M2in a time and dose dependent manner.M2 can secrete more TGF-βand VEGF.Conclusion:1.Energy instrument surgery can promote the proliferation of ovarian cancer cells,and the promotion effect of energy instrument surgery is more obvious than that of non-energy instrument surgery.2.Energy instrument carbonized tissue particles can also promote the proliferation of ovarian cancer cells.It is suggesting that energy instrument surgery can promote the proliferation of ovarian cancer cells more than non-energy instrument surgery is caused by carbonized tissue particles.3.The mechanism of energy instrument carbonized tissue particles promoting ovarian cancer cell proliferation may be as follow:phagocytes(including monocytes and neutrophils)recruitment;antigen presenting cells(including macrophages and dendritic cells)mature and differentiate;Negative immune regulation factors(M2macrophages,regulatory T cells)increased;Various tumor growth-promoting cytokines(TGF-β,VEGF,IFN-γ)increased.Among them,M2 macrophages were the most prominent.4.Macrophage polarization to M2 is an important mechanism of carbonized tissue particles promoting ovarian cancer cell proliferation.(1)Energy instrument inducing carbonized tissue particles can stimulate macrophage recruitment and polarization to M2 in a short time,but the effect is not obvious for a long time(8days and above);But if carbonized tissue particles cooperate with ovarian cancer cells,the pro-tumor effect of M2 can be up-regulated for a long time.(2)Macrophages can be transformed into M2 after being stimulated by carbonized tissue particles in vitro,and there is a time and concentration dependence.(3)Carbonized tissue particles can promote tumor angiogenesis,up-regulate the expression of TGF-βand VEGF,thereby promoting the rapid growth of tumors.5.This study suggests that the use of energy instruments in the surgical treatment of ovarian cancer should try to avoid carbonation tissue,and all visible lesions should be removed.Otherwise,the residual ovarian cancer cell may proliferate after surgery,and the recurrence of ovarian cancer affects the prognosis.