| Objective:Pancreatic cancer(PanCa)is one of the most commonly diagnosed carcinoma,characterized by early lymph node,vascular and liver metastasis. Perineural invasion(PNI)is one of the most important features of pancreatic cancer, and significantly related to the prognosis of the patients.It's believed that PNI is an important risk factor for post-peritoneal recurrence.Little is known about the mechanism of PNI in pancreatic cancer.Ayala et al.showed the utility of the prostate cancer/DRGs in vitro system to study specific mechanism of prostate cancer cell-nerve interaction and perineural invasion.The data suggested that perineural invasion mechanisms involve active and reciprocal interactions between carcinoma cells and adjacent nerve/ganglions in prostate cancer progression.In this study,we presented a novel model system which may facilitate to understand the molecular basis of PNI in pancreatic cancer.The neurite outgrowth,pancreatic cancer cell proliferation and migration were also observed in this model.Methods:Dorsal root ganglia(DRGs)from the cervical,thoracic,and lumbar areas of 4～6 week-old mice were dissected using a posterior approach under sterile conditions. Mouse dorsal root ganglia(DRGs)and human pancreatic cancer cell line(MIAPaca-2) were co-cultured in Matrigel matrix to build this PNI model.At 24h,48h,72h,96h, 120h,and 144h,neurite outgrowth,pancreatic cancer cell colony formation, neurite-colony contact and retrograde migration were observed under inverted microscopy.The data were photographed and quantitated or analyzed with the Image-Pro Plus 5.0 system.The proliferation index(PI)of pancreatic cancer cells was calculated by detecting the positive rate of Ki-67 using immunocytochemistry analysis.The MTT assay was used to detect the absorbance(A)of the pancreatic cancer cells.The apoptotic ratio(AI)was evaluated by flow cytometry analysis.Results:Dorsal root ganglia(DRGs)locate at the ventrospinal where the posterior root of the spinal nerves efferens the spinal cord.It's about 1～2 mm,pale yellow, round,smooth and glossy.Neurite outgrowth was stimulated at the present of pancreatic carcinoma cells with after 72 hours co-culture,which showed about 2.57-fold area larger than that of control(P＜0.001).After 72 hours,MIA PaCa-2 colonies co-cultured with DRGs exhibited 1.33-fold colony area when compared with that of control(P＜0.001).In the co-culture system,neurites exhibited trends of growing towards the pancreatic carcinoma cell colony under the inverted microscopy, and meanwhile,the pancreatic cancer cells migrated to the DRGs along the neurites outgrowth.The positive rate of Ki-67 nuclear antigen was significantly higher than in the co-culture group.The PI value was about 12.804±1.84%(9.8%～15.3%)in the experimental group,while it was about 6.81±0.73%(5.8%～8.3%)in the control, there was statistical difference between the two groups(P＜0.001).The MTT assay showed that the absorbance rate was significantly higher,too,which showed that proliferation of the pancreatic cancer cells is more active than that in the control.Flow cytometry analysis showed that the apoptosis ratio of the pancreatic cancer cell was lower than that of control(2.46±0.37%vs.4.89±0.63%,P＜0.001).Conclusion:In the present study,we construct an in vitro dorsal root ganglia and human pancreatic cancer cell line co-culture model,successfully.In the MIA PaCa-2/DRGs co-culture system,we demonstrate that the neural-pancreatic carcinoma cell interaction which is a mutually beneficial process for the growth of neurites and the pancreatic carcinoma cells.The pancreatic cancer cells showed trends of migrating to the DRGs along the neurites outgrowth.Many cytokines might act synergistically in the process which deserved further investigation.
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