| In China,esophageal squamosal cell carcinoma?ESCC?is the most common pathological type.The recurrence and metastasis of esophageal squamous cell carcinoma caused by chemotherapy resistance is still the main clinical problem.The body's response to chemotherapy will strengthen or weaken the effect of tumor chemotherapy.Immune cells such as macrophages and T cells infiltrating the tumor microenvironment will also activate specific tumor-specific immune responses.The body's immune deficiency may be an important cause of chemotherapy resistance.Therefore,it is of great clinical significance to explore the influence of the body's innate and adaptive immunity on the progression of esophageal squamous cell carcinoma and chemotherapy.NLRP3 inflammasome is a major complex of the body's involvement in the innate immune defense process.It is a protein complex composed of NLRP3 protein receptor,apoptosis-related speck-like protein?ASC?and cysteine aspartate protease precursor?procaspase-1?.Its function makes procaspase-1 self-cleavage into active caspase-1,and cleaves pro-inflammatory cytokines pro-IL-1?and pro-IL-18 into active interleukins IL-1?and IL-18,and exerts corresponding effects.NLRP3inflammasome plays a pro-cancer role in the abnormal activation of a variety of solid tumors such as breast cancer,lung cancer,gastric cancer,melanoma,head and neck squamous cell carcinoma.However,the NLRP3 inflammasome prevents colonic malignant transformation in colon cancer and plays a role in suppressing cancer.However,Lipopolysaccharide?LPS?can activate the NLRP3 inflammasome on Barrett's esophagus and promote the carcinogenesis of Barrett's esophagus clear.Therefore,it is worth further investigation.The study also found that certain chemotherapeutic drugs can activate NLRP3inflammasome in tumor cells and recruit immune cells to infiltrate in the tumor microenvironment,causing changes in the body's tumor immunity to affect the treatment effect.CD47 is a cell surface protein and is the main mechanism of tumor innate immune escape.CD47 can interact with signal regulatory protein??SIRP??expressed on macrophages to send anti-phagocytic signals,preventing macrophages from phagocytosis and killing tumor cells.Our previous research found that human esophageal squamous cell carcinoma not only has high expression of CD47 protein,but also a large number of macrophage infiltration.When CD47 antibody is used to block CD47-SIRP?signal,it can enhance macrophage phagocytosis of esophageal squamous cell carcinoma cells.Therefore,this study proposes a scientific hypothesis:chemotherapy drugs may activate NLRP3 inflammasome of esophageal squamous cell carcinoma to recruit macrophages to the tumor microenvironment,and may lead to treatment resistance through CD47-SIRP?signaling.In summary,in order to explore the role of NLRP3 inflammasome on esophageal squamous cell carcinoma and treatment resistance,the subject is to be derived from human esophageal squamous cell carcinoma tissue specimens,human and mouse esophageal squamous carcinoma cell lines,and wild-type C57BL/6 and C57BL/6-Nlrp3-/-knockout mice animal models were studied at three levels.First,the expression level and clinical significance of NLRP3 inflammasome in human esophageal squamous cell carcinoma tissues are clarified.Secondly,to explore the role and possible mechanism of NLRP3 inflammasome on the proliferation,invasion and migration of esophageal squamous carcinoma cells.Finally,we explored whether NLRP3 inflammasome recruited macrophages and induced cisplatin resistance in tumor-bearing mice through CD47-SIRP?signaling.This experiment aims to provide a theoretical basis for the NLRP3 inflammasome to become a new target for immunotherapy of esophageal squamous cell carcinoma,and to provide a new animal model for studying the immune microenvironment and treatment resistance of esophageal squamous cell carcinoma.This study is divided into the following three parts:Part ? The expression of NLRP3 Inflammasome in Human Esophageal Squamous Cell Carcinoma and Its Clinical SignificanceMethods1.Human esophageal squamous cell carcinoma tissues and clinicopathological characteristics data were collectedIn this study,84 pairs of surgically resected esophageal squamous carcinoma tissues and adjacent tissues were collected as the research object,and the patient's age,gender,depth of primary tumor invasion and TNM stage,lymph node metastasis and distant Transfer information.All patients had no radiotherapy and chemotherapy before surgery.2.The expression levels and clinical significance of NLRP3 inflammasomeThe protein expressions of NLRP3,ASC,caspase-1,IL-1?and Ki67 in esophageal squamous cell carcinoma and adjacent tissues were detected by immunohistochemistry or Western blot.ELISA was used to detect the secretion of IL-1?in esophageal squamous cell carcinoma and adjacent tissues.The correlation between NLRP3 and IL-1?protein expression levels and clinicopathological characteristics and Ki67 was also analyzed.3.The m RNA expression levels of NLRP3 inflammasomeQuantitative real-time PCR?q RT-PCR?was used to detect the m RNA expression of NLRP3,ASC,caspase-1 and IL-1?.Results1.The protein expression of NLRP3,ASC,caspase-1 and IL-1?in esophageal squamous cell carcinoma tissues was elevatedCompared with adjacent tissues,the number of NLRP3,ASC,caspase-1 and IL-1?immunohistochemical staining positive cells in esophageal squamous cell carcinoma tissues increased significantly and was localized in the cytoplasm.Histochemistry score?H-score?showed that the H-score scores of NLRP3,ASC,caspase-1 and IL-1?in esophageal squamous cell carcinoma were significantly higher than those in adjacent tissues?P<0.05?.Western blot also confirmed that the expression of NLRP3,ASC,caspase-1 and IL-1?protein was higher than that of adjacent tissues?P<0.05?.ELISA method found that the concentration of IL-1?in esophageal squamous cell carcinoma was higher than that in adjacent tissues?P=0.001?.2.Correlation between NLRP3 and IL-1?protein expression and clinicopathological characteristics and Ki67The expression of NLRP3 protein was related to the depth of primary tumor invasion and TNM stage,and the difference was statistically significant?P=0.032and P=0.021,respectively?,but not related to the patient's age,gender,lymph node metastasis and distant metastasis?P>0.05?.The expression of IL-1?protein was related to the depth of primary tumor invasion and lymph node metastasis?P<0.05?.Spearman correlation analysis showed that abnormally high expression of NLRP3 in tissues of esophageal squamous cell carcinoma was positively correlated with high expression of Ki67?r=0.355,P=0.014?.3.The m RNA expression of NLRP3,ASC,caspase-1 and IL-1?in esophageal squamous cell carcinomaCompared with adjacent tissues,the proportion of patients whose cancer tissue m RNA expression level increased more than two times was:NLRP3 42.86%?18/42,P=0.009?,ASC 54.76%?23/42,P=0.008?,Caspase-1 accounted for 50.00%?21/42,P=0.003?and IL-1?accounted for 64.28%?27/42,P=0.001?,and there were obvious individual differences in their expression levels.Part ? The effect of the NLRP3 inflammasome on the proliferation,invasion and migration of ESCC cellsMethods1.q RT-PCR and western blot were used to verify the expression of NLRP3 in ESCC cell lines?KYSE70,KYSE510,TE1,TE13,EC9706,EC1,and EC109?and esophageal immortalized cell?Het-1A?.2.Small interfering RNA?si RNA?was used to knockdown the NLRP3 gene in TE13 and KYSE70 cells.At the same time,the NLRP3 overexpressing EC9706 and KYSE510 cell lines were established by constructed pc DNA3.1?+?-NLRP3 vector.3.Based on the above cell models,the cell proliferative capacity was detected by CCK8 method.Cell migration capacity was determined by transwell experiments and scratch assays.The invasion ability was detected by counting transwell cells4.CRISPR/Cas9-mediated gene editing technology was used to establish knock out NLPR3 in TE13 cell,flow cytometry was used to detect cell apoptosis and the expression of E-cadherin and Vimentin was detected by western blot.Results1.Compared with immortalized esophageal epithelial cell line?Het-1A?,the protein expression of NLRP3 were highest in TE13 and KYSE70 cells and the lowest of expression of NLRP3 were in EC9706 and KYSE510 cells?P<0.05?.2.Knockdown of the NLRP3 gene reduced the proliferation capacity of TE13and KYSE70 cells,significantly decreased the migration and invasion ability of TE13and KYSE70 cells,and decreased the healing capacity of wound?P<0.05?.3.Overexpression of the NLRP3 gene enhanced the proliferation of EC9706 and KYSE510 cells,significantly increased the migration and invasion ability of EC9706and KYSE510 cells,increased the healing capacity of wound?P<0.05?.4.TE13 cell line with CRISPR/Cas9 targeted knockout of NLRP3 gene was constructed,the anti-apoptosis ability decreased.The expression of E-cadherin increased and the expression of vimentin decreased,suggesting that epithelial mesenchymal transformation weakened.Part ? NLRP3 Inflammasome Mediates Cisplatin Resistance in Tumor-bearing MiceMethods1.Using wild-type C57BL/6 mice and Nlrp3-/-knockout mice to construct an xenograft tumor model and a cisplatin chemotherapy modelCulture and prepare mouse esophageal squamous carcinoma cell suspension?1×107 cells/ml?.Each mouse was inoculated with 0.1 ml of wild-type C57BL/6 mice and Nlrp3-/-knockout mice to establish a complete immune function in tumor xenograft animal model.When the transplanted tumor volume reached 200 mm3,the tumor-bearing mice were randomly divided into a model group and a cisplatin group.In the cisplatin group,each mouse was given cisplatin at a dose of 2 mg/kg,intraperitoneally injected once a day for 5 days.After 2 days of withdrawal,the drug was continued for 5 days to establish a cisplatin chemotherapy model.The model group was intraperitoneally injected with equal volume of normal saline.Each group draws the growth curve of the transplanted tumor.2.The transplanted tumor tissue cells were cultured in vitro to regenerate wild-type C57BL/6 mice to establish multiple chemotherapy models.Under sterile conditions,the transplanted tumor tissues of mice were harvested,shredded into tissues with a size of about 1 mm3,digested with trypsin for 5 min for a total of 2 times,and then centrifuged to take the precipitate and adhere to the expanded culture.Establish the cisplatin chemotherapy model again by the same method.3.The effect of NLRP3 expression changes on tumor-bearing mice after cisplatin treatmentObserve and compare the growth curve of transplanted tumors of WT and Nlrp3-/-knockout tumor-bearing mice after cisplatin treatment;compare the volume changes of transplanted tumors in tumor-bearing mice before and after multiple cisplatin treatments,and detect the transplantation by immunohistochemistry and Western blot changes of NLRP3 and CD47 protein expression in tumor tissues;ELISA method was used to detect the changes of IL-1?in serum and transplanted tumor tissues of tumor-bearing mice after cisplatin treatment.4.Effects of changes in NLRP3 expression level after cisplatin treatment on macrophage recruitment and CD47-SIRP?signalingThe WT and Nlrp3-/-knockout tumor-bearing mice were used to establish cisplatin chemotherapy models.Immunohistochemical method was used to detect the expression of CD45 and F4/80 protein in the transplanted tumor macrophage markers.Different NLRP3 expression levels and their effects on macrophage recruitment;Western blot was used to detect the expression of SIRP?and CD47 protein in transplanted tumor tissues,and to explore the effect of NLRP3 expression changes on CD47-SIRP?signal after cisplatin treatment.Results1.The effect of NLRP3 expression on the therapeutic effect of cisplatinCompared with Nlrp3-/-knockout,wild-type C57BL/6 mice had a fast-growing tumor and large volume.Cisplatin decreased the tumor inhibition rate of wild-type C57BL/6 mice with statistical difference?P<0.05?.It suggests that NLRP3 expression can promote the growth of transplanted tumors and reduce the effect of cisplatin.2.Repeated cisplatin treatment of wild-type C57BL/6 tumor-bearing mice decreased tumor suppression rate,showing cisplatin resistanceCompared with the tumor-bearing model group?967.9±53.3?mm3,the volume of the first and second administration of cisplatin was significantly reduced,respectively:?423.5±10.3?mm3 and?515.2±40.8?mm3?P<0.05?,And the tumor volume after the third administration was?860.23±24.2?mm3?P>0.05?,which was not significantly different from the model group?P>0.05?.Compared with the tumor weight of the model group,the weight of the first and second cisplatin treatment of transplanted tumors was significantly reduced,the difference was statistically significant?P<0.05?.However,the weight of the transplanted tumor after the third cisplatin treatment was not significantly different from that of the model group.The above results suggest that as the number of cisplatin administrations increases,the tumor inhibition rate decreases,and tumor-bearing mice begin to exhibit cisplatin resistance?P>0.05?.3.Multiple cisplatin treatments increase the expression of NLRP3 protein and IL-1?content in tumor-bearing miceWestern blot analysis of the transplanted tumor tissues of the three-time chemotherapy model found that:compared with the model group,the expression of NLRP3,ASC and caspase-1 protein increased in transplanted tumor tissues of the first cisplatin group,and the second and third cisplatin treatment groups.The protein expression was significantly higher than that in the first cisplatin treatment group?P<0.05?.The mmunohistochemical H-score score of cisplatin group was higher than that of model group?P<0.05?.This suggests that multiple administrations of wild-type tumor-bearing mice with cisplatin can activate the NLRP3 and increase the expression of NLRP3 protein.ELISA results showed that the content of IL-1?in the transplanted tumor tissue was significantly higher than that in the model group?P<0.05?,so cisplatin can activate NLRP3 secretion of IL-1?in transplanted tumor tissue.4.tumor-bearing mice have increased CD47 protein expression after multiple cisplatin treatments,combined with intratumoral injection of CD47antibody can reverse cisplatin resistanceCompared with the model group,the expression of CD47 protein in the transplanted tumor tissues of the cisplatin group increased with the number of cisplatin treatments?P<0.05?.Compared with the cisplatin group alone,the combined use of CD47 antibody intratumoral injection can improve the cisplatin tumor inhibition rate?P<0.05?.5.NLRP3 protein expression can promote the recruitment of macrophages and highly express the role of SIRP?and CD47Compared with Nlrp3-/-knockout tumor-bearing mice,the expression of CD45and F4/80 protein in the transplanted tumor tissue of wild-type C57BL/6 mice was increased,and the expression of CD47 protein and SIPR?protein in the tissues were also increased by Western blot detection?P<0.05?.Compared with the model group,after wild-type C57BL/6 tumor-bearing mice were treated with cisplatin,the expression of CD45 and F4/80 protein in transplanted tumor tissue was significantly increased?P<0.05?,and the expression of CD47protein and SIPR?protein in cisplatin group was also significantly higher than the model group?P<0.05?.However,Nlrp3-/-knockout tumor-bearing mice showed no significant difference in cisplatin treatment?P>0.05?.The above results suggest that cisplatin can promote macrophage infiltration and enhance CD47-SIRP?signal to inhibit macrophage phagocytosis.Conclusions1.The high expression of NLRP3 protein in human esophageal squamous cell carcinoma tissue is related to TNM stage and lymph node metastasis.Down-regulating the expression of NLRP3 protein can inhibit the proliferation,migration and invasion of esophageal squamous carcinoma cells,and its mechanism may be related to the induction of apoptosis and the reduction of epithelial-mesenchymal transition.2.Cisplatin treatment can increase the expression of NLRP3 and CD47 protein in the transplanted tumor tissue of wild-type C57BL/6 mice,reduce the tumor suppression rate,and cause treatment resistance.Cisplatin combined with intratumoral injection of CD47 antibody can reverse treatment resistance.The mechanism may be related to NLRP3 recruiting macrophage infiltration and enhancing SIRP?-CD47 signal. |
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