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Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor prognosis: an in-vivo and in-vitro study.

Vered M, Lehtonen M, Hotakainen L, Pirilä E, Teppo S, Nyberg P, Sormunen R, Zlotogorski-Hurvitz A, Salo T, Dayan D - BMC Cancer (2015)

Bottom Line: RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced.HSC-3-derived exosomes were loaded with CAV1.Accumulation of CAV1-TME in TSCC had a negative prognostic value.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, 69978, Israel. mvered@post.tau.ac.il.

ABSTRACT

Background: Caveolin-1 (CAV1) may be upregulated by hypoxia and acts in a tumor-dependent manner. We investigated CAV1 in tongue squamous cell carcinoma (TSCC) and its association with clinical outcomes, and studied in vitro possible ways for CAV1 accumulation in the tumor microenvironment (TME).

Methods: TSCC cases (N = 64) were immunohistochemically stained for CAV1. Scores were separately assessed in the tumor and TME and plotted for association with recurrence and survival (univariate analysis with log-rank test). In vitro studies were performed on a 3D myoma organotypic model, a mimicker of TME. Prior to monoculturing HSC-3 tongue cancer cells, the model underwent modifications in oxygenation level (1%O2 hypoxia to upregulate CAV1) and/or in the amount of natural soluble factors [deleted by 14-day rinsing (rinsed myoma, RM), to allow only HSC-3-derived factors to act]. Controls included normoxia (21%O2) and naturally occurring soluble factors (intact myoma, IM). HSC-3 cells were also co-cultured with CaDEC12 cells (fibroblasts exposed to human tongue cancer). CAV1 expression and cellular distribution were examined in different cellular components in hypoxic and rinsed myoma assays. Twist served as a marker for the process of epithelial-mesenchymal transition (EMT). Exosomes isolated from HSC-3 media were investigated for containing CAV1.

Results: Expression of CAV1 in TSCC had a higher score in TME than in the tumor cells and a negative impact on recurrence (p = 0.01) and survival (p = 0.003). Monocultures of HSC-3 revealed expression of CAV1 mainly in the TME-like myoma assay, similar to TSCC. CAV1+, alpha-smooth muscle actin (αSMA) + and Twist + CAF-like cells were observed surrounding the invading HSC-3, possibly reflecting EMT. RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced. HSC-3-CaDEC12 co-cultures revealed CAV1+, αSMA+ and cytokeratin-negative CAF-like cells, raising the possibility of CaDEC12 cells gaining a CAF phenotype. HSC-3-derived exosomes were loaded with CAV1.

Conclusions: Accumulation of CAV1-TME in TSCC had a negative prognostic value. In vitro studies showed the presence of CAV1 in cancer cells undergoing EMT and in fibroblasts undergoing trans-differentiation to CAFs. CAV1 delivery to the TME involved cancer cell-derived exosomes.

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HSC-3 co-cultures: intact myoma, normoxia. HSC-3 cells were cocultured with gingival fibroblasts (GFs) or with fibroblasts isolated from a human tongue tumor (called CaDEC12 cells). A few caveolin-1-positive cells are seen in the upper region tumor cells (a, d). Caveolin-1-positive cells are spindle-shaped and concentrically arranged around the invading tumor islands in the lower regions (b, e). Serial sections of d & e (g & h, respectively) of the lower region also show peri-HSC-3 spindle cells positive for alpha-smooth muscle actin (αSMA). Double immunostaining with pan-cytokeratin (AE1/AE3, grey color) and caveolin-1 (brown color) in co-cultures of HSC-3 + GF (c) and HSC-3 + CaDEC12 (f) shows that the spindle-shaped cells surrounding the invading tumor islands are only caveolin-1-positive (black arrows). Some of the cells within the tumor islands are positive for both cytokeratin and caveolin-1 (c, f, red arrows).
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Fig4: HSC-3 co-cultures: intact myoma, normoxia. HSC-3 cells were cocultured with gingival fibroblasts (GFs) or with fibroblasts isolated from a human tongue tumor (called CaDEC12 cells). A few caveolin-1-positive cells are seen in the upper region tumor cells (a, d). Caveolin-1-positive cells are spindle-shaped and concentrically arranged around the invading tumor islands in the lower regions (b, e). Serial sections of d & e (g & h, respectively) of the lower region also show peri-HSC-3 spindle cells positive for alpha-smooth muscle actin (αSMA). Double immunostaining with pan-cytokeratin (AE1/AE3, grey color) and caveolin-1 (brown color) in co-cultures of HSC-3 + GF (c) and HSC-3 + CaDEC12 (f) shows that the spindle-shaped cells surrounding the invading tumor islands are only caveolin-1-positive (black arrows). Some of the cells within the tumor islands are positive for both cytokeratin and caveolin-1 (c, f, red arrows).

Mentions: CAV1 immunoreaction in the HSC-3-CaDEC12 cocultures was low (a score of 1) in the upper region of the myoma in the spindle-shaped, the peri-HSC-3 cells as well as in the HSC-3 cells. The peri-HSC-3 cells in the lower region of the myoma showed a high immunoexpression (a score of 4), whereas the HSC-3 cells demonstrated a low expression (a score of 1) (Figure 4, Table 1). These findings may suggest that a substantial part of the CAV1 is transported from the HSC-3 cells into the spindle cells in the lower region. The expression of CAV1 in the HSC-3-GF co-cultures seemed to be similar in both spindle cells and HSC-3 cells, irrespective of the myoma region. These findings differed from those of the HSC-3-CaDEC12 co-cultures and could be attributed to the different source of the fibroblasts and their crosstalk with the HSC-3 cells. The results of the control HSC-3 monocultures were similar to those of the HSC-3-CaDEC12 cocultures, raising the possibility that introduction of normal GFs could have an effect on the expression of CAV1 within the HSC-3 cells and on its distribution to the surrounding cells within the myoma. Collectively, we suggest that, similarly to other types of carcinomas (e.g., breast, kidney and colon) [13], CAV1 accumulates within the CAFs in tongue cancer as well.Figure 4


Caveolin-1 accumulation in the tongue cancer tumor microenvironment is significantly associated with poor prognosis: an in-vivo and in-vitro study.

Vered M, Lehtonen M, Hotakainen L, Pirilä E, Teppo S, Nyberg P, Sormunen R, Zlotogorski-Hurvitz A, Salo T, Dayan D - BMC Cancer (2015)

HSC-3 co-cultures: intact myoma, normoxia. HSC-3 cells were cocultured with gingival fibroblasts (GFs) or with fibroblasts isolated from a human tongue tumor (called CaDEC12 cells). A few caveolin-1-positive cells are seen in the upper region tumor cells (a, d). Caveolin-1-positive cells are spindle-shaped and concentrically arranged around the invading tumor islands in the lower regions (b, e). Serial sections of d & e (g & h, respectively) of the lower region also show peri-HSC-3 spindle cells positive for alpha-smooth muscle actin (αSMA). Double immunostaining with pan-cytokeratin (AE1/AE3, grey color) and caveolin-1 (brown color) in co-cultures of HSC-3 + GF (c) and HSC-3 + CaDEC12 (f) shows that the spindle-shaped cells surrounding the invading tumor islands are only caveolin-1-positive (black arrows). Some of the cells within the tumor islands are positive for both cytokeratin and caveolin-1 (c, f, red arrows).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4318139&req=5

Fig4: HSC-3 co-cultures: intact myoma, normoxia. HSC-3 cells were cocultured with gingival fibroblasts (GFs) or with fibroblasts isolated from a human tongue tumor (called CaDEC12 cells). A few caveolin-1-positive cells are seen in the upper region tumor cells (a, d). Caveolin-1-positive cells are spindle-shaped and concentrically arranged around the invading tumor islands in the lower regions (b, e). Serial sections of d & e (g & h, respectively) of the lower region also show peri-HSC-3 spindle cells positive for alpha-smooth muscle actin (αSMA). Double immunostaining with pan-cytokeratin (AE1/AE3, grey color) and caveolin-1 (brown color) in co-cultures of HSC-3 + GF (c) and HSC-3 + CaDEC12 (f) shows that the spindle-shaped cells surrounding the invading tumor islands are only caveolin-1-positive (black arrows). Some of the cells within the tumor islands are positive for both cytokeratin and caveolin-1 (c, f, red arrows).
Mentions: CAV1 immunoreaction in the HSC-3-CaDEC12 cocultures was low (a score of 1) in the upper region of the myoma in the spindle-shaped, the peri-HSC-3 cells as well as in the HSC-3 cells. The peri-HSC-3 cells in the lower region of the myoma showed a high immunoexpression (a score of 4), whereas the HSC-3 cells demonstrated a low expression (a score of 1) (Figure 4, Table 1). These findings may suggest that a substantial part of the CAV1 is transported from the HSC-3 cells into the spindle cells in the lower region. The expression of CAV1 in the HSC-3-GF co-cultures seemed to be similar in both spindle cells and HSC-3 cells, irrespective of the myoma region. These findings differed from those of the HSC-3-CaDEC12 co-cultures and could be attributed to the different source of the fibroblasts and their crosstalk with the HSC-3 cells. The results of the control HSC-3 monocultures were similar to those of the HSC-3-CaDEC12 cocultures, raising the possibility that introduction of normal GFs could have an effect on the expression of CAV1 within the HSC-3 cells and on its distribution to the surrounding cells within the myoma. Collectively, we suggest that, similarly to other types of carcinomas (e.g., breast, kidney and colon) [13], CAV1 accumulates within the CAFs in tongue cancer as well.Figure 4

Bottom Line: RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced.HSC-3-derived exosomes were loaded with CAV1.Accumulation of CAV1-TME in TSCC had a negative prognostic value.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Pathology and Oral Medicine, School of Dental Medicine, Tel Aviv University, Tel Aviv, 69978, Israel. mvered@post.tau.ac.il.

ABSTRACT

Background: Caveolin-1 (CAV1) may be upregulated by hypoxia and acts in a tumor-dependent manner. We investigated CAV1 in tongue squamous cell carcinoma (TSCC) and its association with clinical outcomes, and studied in vitro possible ways for CAV1 accumulation in the tumor microenvironment (TME).

Methods: TSCC cases (N = 64) were immunohistochemically stained for CAV1. Scores were separately assessed in the tumor and TME and plotted for association with recurrence and survival (univariate analysis with log-rank test). In vitro studies were performed on a 3D myoma organotypic model, a mimicker of TME. Prior to monoculturing HSC-3 tongue cancer cells, the model underwent modifications in oxygenation level (1%O2 hypoxia to upregulate CAV1) and/or in the amount of natural soluble factors [deleted by 14-day rinsing (rinsed myoma, RM), to allow only HSC-3-derived factors to act]. Controls included normoxia (21%O2) and naturally occurring soluble factors (intact myoma, IM). HSC-3 cells were also co-cultured with CaDEC12 cells (fibroblasts exposed to human tongue cancer). CAV1 expression and cellular distribution were examined in different cellular components in hypoxic and rinsed myoma assays. Twist served as a marker for the process of epithelial-mesenchymal transition (EMT). Exosomes isolated from HSC-3 media were investigated for containing CAV1.

Results: Expression of CAV1 in TSCC had a higher score in TME than in the tumor cells and a negative impact on recurrence (p = 0.01) and survival (p = 0.003). Monocultures of HSC-3 revealed expression of CAV1 mainly in the TME-like myoma assay, similar to TSCC. CAV1+, alpha-smooth muscle actin (αSMA) + and Twist + CAF-like cells were observed surrounding the invading HSC-3, possibly reflecting EMT. RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced. HSC-3-CaDEC12 co-cultures revealed CAV1+, αSMA+ and cytokeratin-negative CAF-like cells, raising the possibility of CaDEC12 cells gaining a CAF phenotype. HSC-3-derived exosomes were loaded with CAV1.

Conclusions: Accumulation of CAV1-TME in TSCC had a negative prognostic value. In vitro studies showed the presence of CAV1 in cancer cells undergoing EMT and in fibroblasts undergoing trans-differentiation to CAFs. CAV1 delivery to the TME involved cancer cell-derived exosomes.

Show MeSH
Related in: MedlinePlus