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Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells.

Mulvey HE, Chang A, Adler J, Del Tatto M, Perez K, Quesenberry PJ, Chatterjee D - BMC Cancer (2015)

Bottom Line: We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis.Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and The Alpert Medical School of Brown University, Coro West, Suite 5.01, One Hoppin St, Providence, RI, 02903, USA. hillary.e.mulvey@gmail.com.

ABSTRACT

Background: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.

Methods: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.

Results: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.

Conclusions: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

Extracellular vesicle-mediated reduction of soft-agar growth. a EVs were isolated from non-malignant (1459) cells. HCT116 cells were co-cultured for 7 days with 1459 EVs. EVs were also isolated from non-malignant patient tissue (004CT Normal and Colo) from 2 patients. HCT116 cells were co-cultured for 7 days with 004CT Normal EVs and Colo EVs, after which time soft agar was cloning was performed. An additional co-culture was set up with HCT116 cells and HCT116 EVs. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. There were 5 dishes/condition. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 + 1459 EVs when compared to untreated HCT116 cells, * p < 0.00001. Decrease in colony formation of HCT116 + 004CT Normal EV compared to untreated HCT116 cells, **p < 0.00001, and HCT116 + Colo EV compared to untreated HCT116 cells, ***p < 0.00001, was also assessed. b Migration assay of HCT cells with and without EV treatment. Migration was measured by counting the number of cells on the filter after 48 h. An increase in migration of HCT116 cells relative to 1459 cells, *p < 0.0001, was analyzed using a paired t-test. Increased migration of 1459+ HCT EV compared to 1459 control cells, **p < 0.0001, was also assessed
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Fig2: Extracellular vesicle-mediated reduction of soft-agar growth. a EVs were isolated from non-malignant (1459) cells. HCT116 cells were co-cultured for 7 days with 1459 EVs. EVs were also isolated from non-malignant patient tissue (004CT Normal and Colo) from 2 patients. HCT116 cells were co-cultured for 7 days with 004CT Normal EVs and Colo EVs, after which time soft agar was cloning was performed. An additional co-culture was set up with HCT116 cells and HCT116 EVs. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. There were 5 dishes/condition. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 + 1459 EVs when compared to untreated HCT116 cells, * p < 0.00001. Decrease in colony formation of HCT116 + 004CT Normal EV compared to untreated HCT116 cells, **p < 0.00001, and HCT116 + Colo EV compared to untreated HCT116 cells, ***p < 0.00001, was also assessed. b Migration assay of HCT cells with and without EV treatment. Migration was measured by counting the number of cells on the filter after 48 h. An increase in migration of HCT116 cells relative to 1459 cells, *p < 0.0001, was analyzed using a paired t-test. Increased migration of 1459+ HCT EV compared to 1459 control cells, **p < 0.0001, was also assessed

Mentions: EVs were harvested from a normal human colon fibroblast cell line (1459), as well as two patient tissue samples of normal colon epithelium (004CT Normal & Colo). Three different co-cultures were set up with malignant human colon tumor cell line HCT116, as follows: HCT116 + 1459 EV, HCT116+ 004CT Normal EV, HCT116 + Colo EV. The number of EVs was also normalized for these experiments (see above). After a 7-day incubation period, each experimental condition was grown in soft agar to measure ability of anchorage independent growth. A reduction in the number of colonies was viewed a shift towards a normal phenotype, reflecting the inability of anchorage-independent growth. The co-cultured cells were grown in soft agar for 14 days. HCT116 cells co-cultured with 1459 EVs displayed a significant decrease (p < 0.00001) in colony formation in comparison to the control HCT116 cells (Fig. 2a). A similar effect was seen from HCT116 cells co-cultured with patient tissue derived vesicles. Co-culture of HCT116 with 004CT Normal EVs and HCT116 with Colo EVs both displayed significant decreases (p < 0.00001, p < 0.00001) in colony formation (Fig. 2a). Our results suggest that EVs isolated from normal colon cells can mediate the reversal of malignant phenotype in colon cancer. An additional co-culture was set up with 1459 cells and EVs isolated from 1459 cells. There was no significant difference between the number of colonies formed by cells from 1459 + 1459 EV co-cultures and 1459 control cells (p = 0.28).Fig. 2


Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells.

Mulvey HE, Chang A, Adler J, Del Tatto M, Perez K, Quesenberry PJ, Chatterjee D - BMC Cancer (2015)

Extracellular vesicle-mediated reduction of soft-agar growth. a EVs were isolated from non-malignant (1459) cells. HCT116 cells were co-cultured for 7 days with 1459 EVs. EVs were also isolated from non-malignant patient tissue (004CT Normal and Colo) from 2 patients. HCT116 cells were co-cultured for 7 days with 004CT Normal EVs and Colo EVs, after which time soft agar was cloning was performed. An additional co-culture was set up with HCT116 cells and HCT116 EVs. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. There were 5 dishes/condition. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 + 1459 EVs when compared to untreated HCT116 cells, * p < 0.00001. Decrease in colony formation of HCT116 + 004CT Normal EV compared to untreated HCT116 cells, **p < 0.00001, and HCT116 + Colo EV compared to untreated HCT116 cells, ***p < 0.00001, was also assessed. b Migration assay of HCT cells with and without EV treatment. Migration was measured by counting the number of cells on the filter after 48 h. An increase in migration of HCT116 cells relative to 1459 cells, *p < 0.0001, was analyzed using a paired t-test. Increased migration of 1459+ HCT EV compared to 1459 control cells, **p < 0.0001, was also assessed
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4522096&req=5

Fig2: Extracellular vesicle-mediated reduction of soft-agar growth. a EVs were isolated from non-malignant (1459) cells. HCT116 cells were co-cultured for 7 days with 1459 EVs. EVs were also isolated from non-malignant patient tissue (004CT Normal and Colo) from 2 patients. HCT116 cells were co-cultured for 7 days with 004CT Normal EVs and Colo EVs, after which time soft agar was cloning was performed. An additional co-culture was set up with HCT116 cells and HCT116 EVs. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. There were 5 dishes/condition. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 + 1459 EVs when compared to untreated HCT116 cells, * p < 0.00001. Decrease in colony formation of HCT116 + 004CT Normal EV compared to untreated HCT116 cells, **p < 0.00001, and HCT116 + Colo EV compared to untreated HCT116 cells, ***p < 0.00001, was also assessed. b Migration assay of HCT cells with and without EV treatment. Migration was measured by counting the number of cells on the filter after 48 h. An increase in migration of HCT116 cells relative to 1459 cells, *p < 0.0001, was analyzed using a paired t-test. Increased migration of 1459+ HCT EV compared to 1459 control cells, **p < 0.0001, was also assessed
Mentions: EVs were harvested from a normal human colon fibroblast cell line (1459), as well as two patient tissue samples of normal colon epithelium (004CT Normal & Colo). Three different co-cultures were set up with malignant human colon tumor cell line HCT116, as follows: HCT116 + 1459 EV, HCT116+ 004CT Normal EV, HCT116 + Colo EV. The number of EVs was also normalized for these experiments (see above). After a 7-day incubation period, each experimental condition was grown in soft agar to measure ability of anchorage independent growth. A reduction in the number of colonies was viewed a shift towards a normal phenotype, reflecting the inability of anchorage-independent growth. The co-cultured cells were grown in soft agar for 14 days. HCT116 cells co-cultured with 1459 EVs displayed a significant decrease (p < 0.00001) in colony formation in comparison to the control HCT116 cells (Fig. 2a). A similar effect was seen from HCT116 cells co-cultured with patient tissue derived vesicles. Co-culture of HCT116 with 004CT Normal EVs and HCT116 with Colo EVs both displayed significant decreases (p < 0.00001, p < 0.00001) in colony formation (Fig. 2a). Our results suggest that EVs isolated from normal colon cells can mediate the reversal of malignant phenotype in colon cancer. An additional co-culture was set up with 1459 cells and EVs isolated from 1459 cells. There was no significant difference between the number of colonies formed by cells from 1459 + 1459 EV co-cultures and 1459 control cells (p = 0.28).Fig. 2

Bottom Line: We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis.Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and The Alpert Medical School of Brown University, Coro West, Suite 5.01, One Hoppin St, Providence, RI, 02903, USA. hillary.e.mulvey@gmail.com.

ABSTRACT

Background: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.

Methods: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.

Results: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.

Conclusions: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus