In breast and ovarian cancer, Akt2 overexpression can regulate the expression of 1 1 in type IV collagen, thereby promoting tumor cell invasion and metastasis (48). stroma are likely to relapse after surgery and have short survival rates (15). However, the functions and mechanisms of FAP-positive PSCs in regulating the malignant behaviors of pancreatic cancer remain largely unknown. Methods Pathological specimens The pathological specimens were obtained from pancreatic cancer patients admitted to the Peking Union Medical College Hospital of Chinese Academy of Medical Sciences. All cases were surgically removed and confirmed by pathological examination. The exclusion criteria were as follows: neoadjuvant treatment, incomplete case data, no follow-up data and difficulty in preparing paraffin specimens for immunization Cytarabine slices required for histochemical staining. This study included 56 patients with pancreatic cancer, including 24 male cases and 32 female patients. Among these patients, 37 were aged <65 years, and 19 were aged >65 years. The tumor site was located in the pancreatic heads of 36 patients and in the Rabbit polyclonal to SUMO4 pancreas body or pancreas tail of 20 cases. Thirty-two cases had low and moderate differentiation, and 24 had high differentiation. Forty-four cases had Tumor Node Metastasis (TNM, 7th edition) stages 1 or 2 2, and 12 had TNM stages 3 or 4 4. Meanwhile, 46 Cytarabine patients underwent R0 resection, and 10 underwent R1 resection. All the patients signed the agreement for scientific research use of the samples. The study was reviewed by the Ethics Committee of Peking Union Medical College Hospital, Chinese Academy of Medical Sciences. Immunohistochemical staining and evaluation Paraffin sections were prepared by screening the pathological tissue sections of cancer tissues and adjacent tissues of pancreatic cancer. The paraffin sections used for immunohistochemistry were dewaxed, subjected to endogenous peroxidase removal, and incubated with primary (rabbit anti human FAP polycolonal antibody, abcam, ab28244; mouse anti human SMA monocolonal antibody, Dako, Clone 1A4) and secondary antibodies (ZSGB-Bio, PV-6001 kit). Following DAB color development, the sections were dehydrated, mounted, and observed under a microscope. Positive expression was indicated by the appearance of brownish-yellow particles in the cytoplasm of the Cytarabine cells. The results of immunohistochemical staining were evaluated by two pathologists who were blinded to the clinicopathological data. Brownish-yellow particles appeared as a marker of positive expression in the cytoplasm of the cells. The FAP expression evaluation criteria were as follows: dyeing area 10% was scored as 0 points; 11% 25% as 1 point; >26% 50% as 2 points; >51% as 3 points. A negative staining intensity was scored as 0 points, poor staining as 1 point, intermediate staining as 2 points, and strong staining as 3 points. The classification of slice staining was divided according to the sum of the stained area and staining intensity score: Cytarabine 3 indicated low expression of FAP (FAP unfavorable, FAP?); >3 indicated high expression of FAP (FAP positive, FAP+) (16). Cell culture The human pancreatic cancer cell lines AsPc1, MIAPaCa2, SW1990, SU86.86, and T3M4 were purchased from the Cell Line Lender of Chinese Academy of Medical Sciences. BxPC3, Capan1, and PANC1 were donated by Professor H. Freiss of the Technical University of Munich, Germany. Nontumor tissues that were surgically removed from pancreatic cancer patients were collected, and human primary PSCs were extracted by the outgrowth method. The isolation and culture of human primary PSCs were performed as described previously (6,17). The original human PSCs were extracted before the 10th generation for subsequent experiments. BxPC3 Cytarabine was cultured in RPMI1640 medium (HyClone, SH30809.01B) containing 10% FBS (HyClone, SH30084.03). AsPc1, MIAPaCa2, SW1990, SU86.86, T3M4, Capan1, PANC1, and human PSCs were cultured in DMEM/high-glucose medium containing 10% FBS (HyClone, SH30022.01B). The cells were cultured at 37 C and 5% CO2. Induction and identification of FAP+ PSCs (a,b)]. The positive expression of FAP protein was mainly.