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Isolation of Fibroblast-Activation Protein-Specific Cancer-Associated Fibroblasts.

ABSTRACT: The current study is to develop a gentle and efficient method for purification of fibroblast-activation protein positive (FAP+) cancer-associated fibroblasts (CAFs) from tumor tissues. Fresh tissues were isolated from BALB/c-Nude mice bearing human liver cancer cell line (HepG2), fully minced and separated into three parts, and digested with trypsin digestion and then treated with collagenase type IV once, twice, or thrice, respectively. Finally, the cells were purified by using FAP magnetic beads. The isolated CAFs were grown in culture medium and detected for the surface expression of fibroblast-activation protein (FAP). The number of adherent cells which were obtained by digestion process with twice collagenase type IV digestion was (5.99 ± 0.18) × 104, much more than that with the only once collagenase type IV digestion (2.58 ± 0.41) × 104 (P < 0.0001) and similar to thrice collagenase type IV digestion. The percentage of FAP+ CAFs with twice collagenase type IV digestion (38.5%) was higher than that with the only once collagenase type IV digestion (20.0%) and little higher than thrice collagenase type IV digestion (37.5%). The FAP expression of CAFs was quite different from normal fibroblasts (NFs). The fibroblasts isolated by the innovation are with high purity and being in wonderful condition and display the features of CAFs.

SUBMITTER: Huang Y

PROVIDER: S-EPMC5584363 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Isolation of Fibroblast-Activation Protein-Specific Cancer-Associated Fibroblasts.

Huang Yingying Y Zhou Sufang S Huang Yong Y Zheng Duo D Mao Qiqi Q He Jian J Wang Yiwei Y Xue Dabing D Lu Xiaoling X Yang Nuo N Zhao Yongxiang Y

BioMed research international 20170814

The current study is to develop a gentle and efficient method for purification of fibroblast-activation protein positive (FAP<sup>+</sup>) cancer-associated fibroblasts (CAFs) from tumor tissues. Fresh tissues were isolated from BALB/c-Nude mice bearing human liver cancer cell line (HepG2), fully minced and separated into three parts, and digested with trypsin digestion and then treated with collagenase type IV once, twice, or thrice, respectively. Finally, the cells were purified by using FAP m ...[more]

PMID: 28890895

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Project description:BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive desmoplastic stromal response. Fibroblast activation protein-α (FAP) is best known for its presence in stromal cancer-associated fibroblasts (CAFs). Our aim was to assess whether FAP expression was associated with the prognosis of patients with PDAC and to investigate how FAP expressing CAFs contribute to the progression of PDAC.MethodsFAP expression was immunohistochemically assessed in 48 PDAC specimens. We also generated a fibroblastic cell line stably expressing FAP, and examined the effect of FAP-expressing fibroblasts on invasiveness and the cell cycle in MiaPaCa-2 cells (a pancreatic cancer cell line).ResultsStromal FAP expression was detected in 98% (47/48) of the specimens of PDAC, with the intensity being weak in 16, moderate in 19, and strong in 12 specimens, but was not detected in the 3 control noncancerous pancreatic specimens. Patients with moderate or strong FAP expression had significantly lower cumulative survival rates than those with negative or weak FAP expression (mean survival time; 352 vs. 497 days, P = 0.006). Multivariate analysis identified moderate to strong expression of FAP as one of the factors associated with the prognosis in patients with PDAC. The intensity of stromal FAP expression was also positively correlated to the histological differentiation of PDAC (P < 0.05). FAP-expressing fibroblasts promoted the invasiveness of MiaPaCa-2 cells more intensively than fibroblasts not expressing FAP. Coculture with FAP-expressing fibroblasts significantly activated cell cycle shift in MiaPaCa-2 cells compared to coculture with fibroblasts not expressing FAP. Furthermore, coculture with FAP expressing fibroblasts inactivated retinoblastoma (Rb) protein, an inhibitor of cell cycle progression, in MiaPaCa-2 cells by promoting phosphorylation of Rb.ConclusionsThe present in vitro results and the association of FAP expression with clinical outcomes provide us with a better understanding of the effect of FAP-expressing CAFs on the progression of PDAC.

Dataset Information

Isolation of Fibroblast-Activation Protein-Specific Cancer-Associated Fibroblasts.

Publications

Isolation of Fibroblast-Activation Protein-Specific Cancer-Associated Fibroblasts.

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