| BackgroundPancreatic cancer is a malignant condition of the gastrointestinal tract with extremely high malignancy and poor prognosis.The most common type of pancreatic malignancy is pancreatic ductal adenocarcinoma.Because of lacking specific symptoms and effective therapies,pancreatic cancer has always been one of the most serious challenges in digestive tumors.Clinical and epidemiological studies suggest that smoking,alcohol,genetic factors,pancreatitis,and infection are risk factors for pancreatic cancer.The mechanism of pancreatic carcinogenesis needs to be studied in animal models to investigate the molecular and cellular biological features,which is undoubtedly an important basis for solving clinical problems.Whether pancreatic ductal adenocarcinoma,the main pathological type of pancreatic cancer,originate from pancreatic ductal cells? Different original cells may affect their various biological behavior for tumor formation.Inflammation is an important tumor promoter,but the characteristics of inflammation that promote the development of pancreatic cancer are not clear.Gene mutation is the key event of tumorigenesis,and KRAS,TP53,CDKN2 A and CDKN2 B are the common mutated genes in pancreatic cancer.However,different combinations of carcinogenic mutations may be through multiple mechanisms(autophagy,cell cycle,differentiation,etc.)to contribute to pancreatic cancer development and also make the tumor heterogeneous,which has become a real challenge in clinical practice.ObjectiveThis study aimed to establish different types of pancreatic cancer models in adult animals to investigate the cancer cell origin,pathological characteristics,and pathophysiological mechanisms of pancreatic cancer.Exploring the role of mutated genes in the development of pancreatic cancer from the perspective of inflammation and autophagy,and looking for new possible therapeutic strategies for pancreatic cancer.MethodsIn vivo study,9-week-old male mice were used for experiments.Thirty-five C57BL/6 mice were injected p Tomo-H1-sh Tp53-CMV-KRASG12D-IRESEGFP-U6-sh Cdkn2a/sh Cdkn2 b lentivirus into the head of the pancreas.Fifteen of them were sacrificed at different time points(day 3,day 4,day 7,day 14 and day 21 after injection)to assess the pancreatic pathological features.Twenty mice were randomly assigned to the control and HCQ groups.HCQ mice received hydroxychloroquine(HCQ)(60mg/kg/every two days)by gastric gavage and control mice received saline(100μl/every two days).When the mice were euthanized to minimize their suffering,they were sacrificed by cervical dislocation.Thirty six C57BL/6 mice were randomly divided into four groups to be injected with different lentivirus(p Tomo-H1-sh Tp53-CMV-KRASG12D-IRESEGFP(KP),p Tomo-H1-sh Tp53-CMV-KRASG12DIRESEGFP-U6-sh Cdkn2a(KP-2A),p Tomo-H1-sh Tp53-CMV-KRASG12DIRESEGFP-U6-sh Cdkn2b(KP-2B)and p Tomo-H1-sh Tp53-CMV-KRASG12DIRESEGFP-U6-sh Cdkn2a/sh Cdkn2b(KP-2AB))and were sacrificed at different time points(day 7,day 14 and day 21 after injection).Fifty adult C57 BL / 6 mice were transfected into the pancreatic head by electroporation using KRASG12D-shp53-containing plasmids,of which 18 mice were taken to observe the pathophysiological characteristics of pancreatic tissue at day 1,day 3,day 4,day 7,day 14 and day 21 after transfection.The remaining 32 mice were randomly assigned to the control and celecoxib groups.The control group was given intragastric saline(100μl,once daily),the celecoxib group was given celecoxib intragastrically(60 mg/kg,once daily)after the first day of modeling.6 mice from each group were randomly sampled at day 7and day 14 after modeling,and the remaining mice were sacrificed by cervical dislocation when they were unable to take in food and water.Twelve adult tree shrews were injected with lentivirus containing p Tomo-H1-sh Tp53-CMV-KRASG12DIRESEGFP-U6-sh Cdkn2a/sh Cdkn2 b into their pancreatic head,of which 9 tree shrews were sampled at day 4,day 7 and day 14 after injection,and the remaining mice were sacrificed by cervical dislocation when they were unable to take in food and water.Pancreas tissues were divided into 3 parts from all animals: one part was fixed with 4% paraformaldehyde,sectioned after paraffin embedding,and stained with HE and immunofluorescence(IF);one part was fixed and wrapped with 2.5%glutaraldehyde and the ultrathin sections were buried and examined by transmission electron microscopy;one part was immediately frozen in liquid nitrogen and frozen to-80 ℃ for real-time PCR(RT-PCR)and Western Blot.The pathophysiological changes of pancreas tissues in different pancreatic cancer animal models were observed by HE staining;Pancreatic expressions of pancreatic ductal cells(CK-19),pancreatic acinar cells(CPA1),fluorescent tag(EGFP),neutrophil Cells(MPO),T cells(CD3),B cells(Pax5),macrophages(F4 / 80),nuclear factor kappa-light-chainenhancer of activated B cells(NF-κB),cyclooxygenase-2(COX-2),cell proliferation(Ki67),autophagosome(LC3),autophagy-related genes and proteins(Atg12-Atg5 complex,Atg7 and Atg5),autophagy-related regulatory proteins(CDK5),lysosomal membrane markers(LAMP1 and LAMP2),LC3 substrate(P62),phosphorylated retinoblastoma protein(p Rb1)were analyzed by RT-PCR,IF or Western Blot.Transmission electron microscopy was used to observe the existence of autophagosomes(bilayer membrane structure)in pancreatic tumor cells.The survival curves were used to assess carcinogenesis effects and survival of different pancreatic cancer animal models.In vitro study,we used different lentivirus(p Tomo-H1-sh Tp53-CMVKRASG12D-IRESEGFP(KP),p Tomo-H1-sh Tp53-CMV-KRASG12D-IRES EGFP-U6-sh Cdkn2a(KP-2A),p Tomo-H1-sh Tp53-CMV-KRASG12D-IRESEGFP-U6-sh Cdk2b(KP-2B)and p Tomo-H1-sh Tp53-CMV-KRASG12D-IRESEGFP-U6-sh Cdkn2a/sh Cdkn2b(KP-2AB))to infect mouse pancreatic duct cells.The expressions of autophagosome(LC3)and fluorescent tag(EGFP)were detected by IF.Protein expressions of autophagosome(LC3),autophagy-related proteins(Atg12-Atg5 complex and Atg7),autophagy-related regulatory proteins(CDK5),lysosomal membrane markers(LAMP1 and LAMP2),and LC3 substrate(P62)were detected by Western Blot.Results1.Successfully constructed three different adult animal pancreatic cancer models(lentiviral mouse and lentiviral tree shrew and electroporation mouse models),with a 100% success rate.The pathological features of tumors caused by the three models were similar to undifferentiated carcinomas of pancreatic ductal adenocarcinoma.The mostly infected cells of the three models were all pancreatic epithelial cells,of which the lentiviral mouse model was ductal cell,and the lentiviral tree shrew model and the electroporation mouse model were acinar cells.2.Electroporation resulted in inflammation of pancreas in mice: At day 7 and day 14 after electroporation,HE staining showed a large number of inflammatory cells infiltrated.The expressions of 4 types of inflammatory cells in pancreas of the electroporation mouse group were significantly higher than that in the lentivirus mouse model group,MPO(0.16 ± 0.01 vs 0.05 ± 0.01,p= 0.0002),CD3(0.08 ± 0.02 vs 0.03 ± 0.01,p= 0.0328),Pax5(0.08 ± 0.01 vs 0.05 ± 0.01,p= 0.0393),F4/80(0.04 ± 0.01 vs 0.01 ± 0.01,p= 0.0108),and the sharp increase in neutrophils is the primary manifestation in electroporation mouse group(at day 14 of modeling).The expression of COX-2 in the pancreas tissues of the electroporation model group was significantly higher than that of the lentivirus model group and normal control group(both p<0.0001);In addition,the expression of NF-κB in electroporation model group was also significantly higher than that of the lentivirus model group and the normal control group(both p<0.0001).3.Inflammation of pancreas in the early stage of electroporation: On the first day after electroporation,a large amount of inflammatory cells infiltration was observed in the pancreatic tissue.IF showed that the inflammation infiltration was mainly composed of neutrophil(MPO),and on the third day the number of T cells(CD3)increased.4.Electroporation promoted proliferation and metaplasia of mouse pancreatic acinar cells: On the 7th day after electroporation,the expressions of Ki67 in lentivirus model group and the electroporation model group were significantly higher than that of the normal control group(both p<0.01).Besides,the expression of Ki67 in electroporation group was significantly higher than that of the lentivirus group(p= 0.0002).The co-expressions of CPA1(acinar cell molecular marker)and CK-19(ductal cell molecular marker)were observed in pancreatic tissue on day 7 and day 14 after electroporation.5.Celecoxib prolonged the survival of electroporation model mice: On day 7 after electroporation,the expressions of 4 types of inflammatory cells in the celecoxib group(except F4 / 80)were significantly lower than that of control group(MPO 0.09 ± 0.01 vs 0.19 ± 0.03,p= 0.0214;CD3 0.10 ± 0.01 vs 0.28 ± 0.017,p<0.0001;Pax5 0.08 ± 0.004 vs 0.16 ± 0.02,p= 0.0025;F4 / 80 0.01 ± 0.0005 vs 0.01 ± 0.001,p> 0.05).The survival curve showed that celecoxib could significantly prolong the survival of electroporation model mice(p= 0.0022).6.The level of autophagy increased in lentivirus mice model: The expression levels of autophagy related genes(Atg5,Atg7 and LAMP1)in pancreatic cancer of lentivirus model mice were significantly higher than that of normal mice pancreas(all p<0.05).The expressions of LC3 and LAMP1 in tumor tissues were significantly higher than that of normal mouse pancreas,and the co-expressions of LC3 and LAMP1 were detected in the tumor tissue.Under the transmission electron microscope,there were some double-layered membrane structure in tumor cells.On day 7,day 14,and day 21 after lentivirus infection,the percentage of LC3/EGFP was increased with time,and the proportion on day 21 was 7.4 times of day 7(p<0.05).7.The level of autophagy increased in the pancreatic duct cells infected by lentivirus: After lentivirus infection,the positive expression of LC3 significantly increased in the experimental group(p= 0.0104).Western Blot showed that the expressions of autophagy-related proteins(Atg12-Atg5 protein complex and Atg7),autophagy-related regulatory proteins(CDK5),lysosomal membrane(LAMP1 and LAMP2),and LC3 substrate(P62)increased in experimental group.8.PDAC formation could be significantly decelerated by inhibiting autophagy: Lentiviral mouse model was treated with HCQ and found PDAC formation was significantly decelerated.The median survival time of the HCQ group was significantly longer than that of the control group(70 days vs 57 days,p= 0.0036).Atg5 or Atg7 conditional knockout mice were used to construct pancreatic cancer models by using lentiviral vectors.The median survival time of Atg5loxp/loxp mice(77 days)and Atg7loxp/loxp mice(70.5 days)was significantly longer than that of Atg5+/+ mice(57 days)and Atg7+/+ mice(57 days)(all p<0.05).The expressions of LC3 in Atg5+/+ and Atg7+/+ mice tumor tissue were significantly higher than that of the Atg5 loxp / loxp and Atg7loxp/loxp mice tumor tissue(both p<0.0001).9.The levels of inflammation and autophagy in lentiviral tree shrew model: On day 7 after lentivirus injection,there was no obvious inflammation in the pancreas.On day 14,there have been tiny tumors in the pancreas and the infiltration of inflammatory cells was obvious in tumor area.However,there was no obvious inflammatory cells in the para cancerous tissues.The expressions of LC3 in pancreas tissue on day 7 and day 14 were significantly higher than that of normal tree shrew.10.Levels of autophagy were diverse depend on different models: The expression of LC3 in the pancreatic cancer of lentivirus mouse group was 7 times higher than that of the electroporation mouse group(p<0.0001).The expression of LC3 detected by Western Blot was consistent with the observation of IF.On day 7,day 14,and day 21 after electroporation,the expression of LC3 was 0.136 ± 0.007,0.090 ± 0.008 and 0.041 ± 0.004(all p<0.05).The expressions of LC3 were increased in lentivirus mouse model and lentiviral tree shrew model compared with electroporation mouse model(both p<0.0001).11.Gene screening for increasing autophagy activity: In vivo,the expression of LC3 in pancreatic cancer tissue of 2AB group was higher than that of the KP,KP-2A and KP-2B groups.In vitro,the KP,KP-2A and KP-2B group cells showed a weak expression of LC3,while the KP-2AB group showed stronger expression of LC3(all p<0.05).The autophagy-related proteins(LC3,Atg12-Atg5 protein complex,Atg7 and P62)of each group were assessed by Western Blot,and the results suggested that the autophagy activity of the KP-2AB group was significantly higher than the other three groups.Furthermore,the expression of p Rb1 in normal mouse pancreas and electroporation mouse model were lower than lentiviral mouse model(both p<0.0001).ConclusionThe heterogeneity of the originating cells may be part of the complex carcinogenic mechanism of pancreatic cancer.By increasing the expressions of NF-κB and COX-2,active inflammation which characterized by neutrophil infiltration induced acinar ductal metaplasia.Thereby,the inflammation synergized with a small number of mutated genes to amplify its tumorigenic effect and promote the occurrence of pancreatic cancer.Such pancreatic inflammation should be regarded as a high-risk inflammation associated with pancreatic cancer and celecoxib,the selective COX-2 inhibitor,could prolong the time of tumor formation by reducing this inflammation.Additionally,altering Cdkn2 a and Cdkn2 b genes with the mutations of KRASG12 D and TP53 in pancreas tissue could increase the level of autophagy.The treatment of autophagy inhibitor(HCQ)might be a potentially successful strategy for pancreatic cancer.However,the level of autophagy was low in pancreatic cancer tissue when only involved KRASG12 D and TP53 mutations,and there was no basis for the treatment of autophagy inhibitors.Although autophagy and inflammation both promoted the development of pancreatic cancer,they had different cell-biological mechanisms.Combining diagnostic genetic testing with the treatments of autophagy inhibitors and COX-2 inhibitors may benefit for the clinical strategy of pancreatic cancer. |
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