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

Huang, Yingying; Zhou, Sufang; Huang, Yong; Zheng, Duo; Mao, Qinan; He, Jian; Wang, Yiwei; Xue, Dabing; Lü, Xiaoling; Yang, Nuo; Zhao, Yongxiang

doi:10.1155/2017/4825108

Cited by 18 publications

(17 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Collagenase type I is widely used in digest protocols and cell culture due to its protease potential for disassembling collagen fibrils from the connective tissue. Furthermore, collagenase type I led to the isolation of fibroblasts [ 33 ] and the culture of small intestinal epithelial cells [ 34 ]. Dispase II is a collagenase type IV with a mild proteolytic activity that has also been used in animal tissues for isolating labile primary cells such as porcine urothelial cells, lymphatic and embryonic endothelial cells, stem cells and Schwann cells [ 35 – 38 ].…”

Section: Discussionmentioning

confidence: 99%

A novel method for isolation and culture of primary swine gastric epithelial cells

Bautista-Amorocho

Silva-Sayago

Goyeneche-Patino

et al. 2021

BMC Mol and Cell Biol

View full text Add to dashboard Cite

Background Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach. Results Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively. Conclusions A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

show abstract

Section: Discussionmentioning

confidence: 99%

A novel method for isolation and culture of primary swine gastric epithelial cells

Bautista-Amorocho

Silva-Sayago

Goyeneche-Patino

et al. 2021

BMC Mol and Cell Biol

View full text Add to dashboard Cite

show abstract

“…CAFs constitute the majority of noncancerous cells within a tumor and share many similarities with myofibroblasts found during wound heal-ing. They express, among other things, a-smooth muscle actin (aSMA), fibroblast-activated protein (FAP), and Galectin (3,4). CAFs arise from nearby healthy fibroblasts (HF) that have been differentiated under the tumor microenvironment conditions, and they contribute to the formation of cancer-altered stroma (5,6).…”

Section: Introductionmentioning

confidence: 99%

Exosomes Induce Fibroblast Differentiation into Cancer-Associated Fibroblasts through TGFβ Signaling

Goulet

Bernard

Tremblay³

et al. 2018

Molecular Cancer Research

236

145

View full text Add to dashboard Cite

A particularly important tumor microenvironment relationship exists between cancer cells and surrounding stromal cells. Fibroblasts, in response to cancer cells, become activated and exhibit myofibroblastic characteristics that favor invasive growth and metastasis. However, the mechanism by which cancer cells promote activation of healthy fibroblasts into cancer-associated fibroblasts (CAF) is still not well understood. Exosomes are nanometer-sized vesicles that shuttle proteins and nucleic acids between cells to establish intercellular communication. Here, bladder cancer-derived exosomes were investigated to determine their role in the activation of healthy primary vesical fibroblasts. Exosomes released by bladder cancer cells are internalized by fibroblasts and promoted the proliferation and expression of CAF markers. In addition, cancer cell-derived exosomes contain TGFβ and in exosome-induced CAFs SMAD-dependent signaling is activated. Furthermore, TGFβ inhibitors attenuated CAF marker expression in healthy fibroblasts. Therefore, these data demonstrate that bladder cancer cells trigger the differentiation of fibroblasts to CAFs by exosomes-mediated TGFβ transfer and SMAD pathway activation. Finally, exosomal TGFβ localized inside the vesicle and contributes 53.4% to 86.3% of the total TGFβ present in the cancer cell supernatant. This study highlights a new function for bladder cancer exosomes as novel modulators of stromal cell differentiation. This study identifies exosomal TGFβ as new molecular mechanism involved in cancer-associated fibroblast activation. .

show abstract

“…Collagenase type I is widely used in digest protocols and cell culture due to its protease potential for disassembling collagen brils from the connective tissue. Furthermore, collagenase type I led to the isolation of broblasts [31] and the culture of small intestinal epithelial cells [32]. Dispase II is a collagenase type IV with a mild proteolytic activity that has also been used in animal tissues for isolating labile primary cells such as porcine urothelial cells, lymphatic and embryonic endothelial cells, stem cells and Schwann cells [33][34][35][36].…”

Section: Discussionmentioning

confidence: 99%

A novel method for isolation and culture of primary swine gastric epithelial cells

Bautista-Amorocho

Silva-Sayago

Goyeneche-Patino

et al. 2021

Preprint

View full text Add to dashboard Cite

Background: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.Results: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.Conclusions: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

show abstract

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

Cited by 18 publications

References 28 publications

A novel method for isolation and culture of primary swine gastric epithelial cells

A novel method for isolation and culture of primary swine gastric epithelial cells

Exosomes Induce Fibroblast Differentiation into Cancer-Associated Fibroblasts through TGFβ Signaling

A novel method for isolation and culture of primary swine gastric epithelial cells

Contact Info

Product

Resources

About