Limits...
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 induction of soft-agar growth. EVs were isolated from malignant (HCT116) cells and from patient tumor tissue (004CT Tumor). 1459 cells were co-cultured for 7 days with HCT116 EVs and 004CT Tumor EVs. In both experiments, cells were harvested and utilized for soft agar assay. 1459 cells were also co-cultured with EVs isolated from 1459 cells. Soft agar cloning was performed for 2 weeks and cell colonies were counted with 5 fields/dish using the 40× objective. There were 5 dishes/condition. To provide an estimation of colony size that was evaluated, the area for the colonies counted in an average field was determined. The average area (μm2) determined were: 1459 CTRL, 1988; 1459 + 1459EV, 2603; 1459 + HCT EV, 1860; 1459 + 004CT Tumor EV, 3841. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. A paired t-test was performed to analyze the increase in soft agar colony formation of 1459 + HCT116 EVs when compared to untreated 1459 cells, *p < 0.00001. Increase in colony formation of 1459 + 004CT Tumor EV compared to untreated 1459 cells, **p < 0.00001, was also assessed
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Extracellular vesicle-mediated induction of soft-agar growth. EVs were isolated from malignant (HCT116) cells and from patient tumor tissue (004CT Tumor). 1459 cells were co-cultured for 7 days with HCT116 EVs and 004CT Tumor EVs. In both experiments, cells were harvested and utilized for soft agar assay. 1459 cells were also co-cultured with EVs isolated from 1459 cells. Soft agar cloning was performed for 2 weeks and cell colonies were counted with 5 fields/dish using the 40× objective. There were 5 dishes/condition. To provide an estimation of colony size that was evaluated, the area for the colonies counted in an average field was determined. The average area (μm2) determined were: 1459 CTRL, 1988; 1459 + 1459EV, 2603; 1459 + HCT EV, 1860; 1459 + 004CT Tumor EV, 3841. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. A paired t-test was performed to analyze the increase in soft agar colony formation of 1459 + HCT116 EVs when compared to untreated 1459 cells, *p < 0.00001. Increase in colony formation of 1459 + 004CT Tumor EV compared to untreated 1459 cells, **p < 0.00001, was also assessed

Mentions: Anchorage independent growth is a major hallmark of malignant cells; therefore, we quantified EV-mediated phenotype changes in malignant colon cells using soft agar colony formation. EVs were harvested from a malignant human colon tumor cell line (HCT116) and from a patient tissue sample of malignant colon tumor (004CT Tumor). Two co-cultures were prepared with normal human colon fibroblast cell line 1459: 1459 + HCT116 EVs and 1459 + 004CT Tumor EVs. After a 7-day period of culture, each experimental condition was grown in soft agar to measure ability of anchorage independent growth. Malignancy includes increased anchorage-independent growth, so an increase in the number of colonies was viewed as a shift towards malignant phenotype. 1459 cells co-cultured with HCT116 EVs displayed a significant increase (p < 0.00001) in colony formation in comparison to the 1459 control cells (Fig. 1). The co-culture of 1459 and patient derived 004CT Tumor EVs also displayed a significant increase (p < 0.000001) in colony formation, suggesting an induction of malignant phenotype by EVs isolated from malignant colon tumor cells (Fig. 1). EVs from HCT116 cells were also co-cultured with HCT116 cells to determine if vesicles impact their own cells. There was no significant change in the number of colonies formed by the HCT116 + HCT116 EV co-culture, compared to HCT control (p = 0.33).Fig. 1


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 induction of soft-agar growth. EVs were isolated from malignant (HCT116) cells and from patient tumor tissue (004CT Tumor). 1459 cells were co-cultured for 7 days with HCT116 EVs and 004CT Tumor EVs. In both experiments, cells were harvested and utilized for soft agar assay. 1459 cells were also co-cultured with EVs isolated from 1459 cells. Soft agar cloning was performed for 2 weeks and cell colonies were counted with 5 fields/dish using the 40× objective. There were 5 dishes/condition. To provide an estimation of colony size that was evaluated, the area for the colonies counted in an average field was determined. The average area (μm2) determined were: 1459 CTRL, 1988; 1459 + 1459EV, 2603; 1459 + HCT EV, 1860; 1459 + 004CT Tumor EV, 3841. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. A paired t-test was performed to analyze the increase in soft agar colony formation of 1459 + HCT116 EVs when compared to untreated 1459 cells, *p < 0.00001. Increase in colony formation of 1459 + 004CT Tumor EV compared to untreated 1459 cells, **p < 0.00001, was also assessed
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Extracellular vesicle-mediated induction of soft-agar growth. EVs were isolated from malignant (HCT116) cells and from patient tumor tissue (004CT Tumor). 1459 cells were co-cultured for 7 days with HCT116 EVs and 004CT Tumor EVs. In both experiments, cells were harvested and utilized for soft agar assay. 1459 cells were also co-cultured with EVs isolated from 1459 cells. Soft agar cloning was performed for 2 weeks and cell colonies were counted with 5 fields/dish using the 40× objective. There were 5 dishes/condition. To provide an estimation of colony size that was evaluated, the area for the colonies counted in an average field was determined. The average area (μm2) determined were: 1459 CTRL, 1988; 1459 + 1459EV, 2603; 1459 + HCT EV, 1860; 1459 + 004CT Tumor EV, 3841. The data represents the mean +/− s.d. of 2 independent experiments performed in triplicate. A paired t-test was performed to analyze the increase in soft agar colony formation of 1459 + HCT116 EVs when compared to untreated 1459 cells, *p < 0.00001. Increase in colony formation of 1459 + 004CT Tumor EV compared to untreated 1459 cells, **p < 0.00001, was also assessed
Mentions: Anchorage independent growth is a major hallmark of malignant cells; therefore, we quantified EV-mediated phenotype changes in malignant colon cells using soft agar colony formation. EVs were harvested from a malignant human colon tumor cell line (HCT116) and from a patient tissue sample of malignant colon tumor (004CT Tumor). Two co-cultures were prepared with normal human colon fibroblast cell line 1459: 1459 + HCT116 EVs and 1459 + 004CT Tumor EVs. After a 7-day period of culture, each experimental condition was grown in soft agar to measure ability of anchorage independent growth. Malignancy includes increased anchorage-independent growth, so an increase in the number of colonies was viewed as a shift towards malignant phenotype. 1459 cells co-cultured with HCT116 EVs displayed a significant increase (p < 0.00001) in colony formation in comparison to the 1459 control cells (Fig. 1). The co-culture of 1459 and patient derived 004CT Tumor EVs also displayed a significant increase (p < 0.000001) in colony formation, suggesting an induction of malignant phenotype by EVs isolated from malignant colon tumor cells (Fig. 1). EVs from HCT116 cells were also co-cultured with HCT116 cells to determine if vesicles impact their own cells. There was no significant change in the number of colonies formed by the HCT116 + HCT116 EV co-culture, compared to HCT control (p = 0.33).Fig. 1

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