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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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Caveolin-1 expression in HSC-3 cell myoma monocultures. A. Western blotting: caveolin-1 is detected as complex forms in intact myoma (IM) tissue disc extracts (without added cells), and, to a lesser degree, in the rinsed myoma (RM, without added cells and without soluble factors) disc extracts. Monomeric caveolin-1 is absent from both IM and RM tissue extracts. B. HSC-3 monocultures within rinsed myoma (RM) in both normoxic and hypoxic conditions (a, b) show delicate, spindle, caveolin-1-positive cells closely surrounding invading HSC-3 islands. Alpha-smooth muscle actin immunostaining in normoxic and hypoxic conditions (c, d) shows findings similar to caveolin-1 in terms of cell distribution and morphology. HSC-3 monocultures within intact myoma (IM) immunostained with caveolin-1 (e, f) and alpha-smooth muscle (g, h) show findings similar to those of the RM, respectively. CAV1 and alpha-smooth muscle actin positively stain the spindle cells surrounding the invading HSC-3 tumor islands (indicated by arrows). In all sections, the HSC-3 cells are only occasionally CAV1-positive in both RM and IM, and in both normoxia and hypoxia. However, the spindle cells are usually CAV1-positive, showing a high staining intensity.
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Fig2: Caveolin-1 expression in HSC-3 cell myoma monocultures. A. Western blotting: caveolin-1 is detected as complex forms in intact myoma (IM) tissue disc extracts (without added cells), and, to a lesser degree, in the rinsed myoma (RM, without added cells and without soluble factors) disc extracts. Monomeric caveolin-1 is absent from both IM and RM tissue extracts. B. HSC-3 monocultures within rinsed myoma (RM) in both normoxic and hypoxic conditions (a, b) show delicate, spindle, caveolin-1-positive cells closely surrounding invading HSC-3 islands. Alpha-smooth muscle actin immunostaining in normoxic and hypoxic conditions (c, d) shows findings similar to caveolin-1 in terms of cell distribution and morphology. HSC-3 monocultures within intact myoma (IM) immunostained with caveolin-1 (e, f) and alpha-smooth muscle (g, h) show findings similar to those of the RM, respectively. CAV1 and alpha-smooth muscle actin positively stain the spindle cells surrounding the invading HSC-3 tumor islands (indicated by arrows). In all sections, the HSC-3 cells are only occasionally CAV1-positive in both RM and IM, and in both normoxia and hypoxia. However, the spindle cells are usually CAV1-positive, showing a high staining intensity.

Mentions: Although cells within the myoma discs are no longer expected to be viable [19], the microenvironment is rich in soluble factors with necessary functions for facilitating tumor growth and invasion. Depletion of these factors (including CAV1) by rinsing the myoma discs approached a state in which factors released into the myoma assay originated from the cultured HSC-3 cells. Western blotting of either RM or IM discs showed no monomeric 21 kDa form of CAV1 but only complex forms of immunoreactants (Figure 2A). Thus, it can be inferred that CAV1 in the myoma model is expected to originate from the cell lines cultured in this assay.Figure 2


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)

Caveolin-1 expression in HSC-3 cell myoma monocultures. A. Western blotting: caveolin-1 is detected as complex forms in intact myoma (IM) tissue disc extracts (without added cells), and, to a lesser degree, in the rinsed myoma (RM, without added cells and without soluble factors) disc extracts. Monomeric caveolin-1 is absent from both IM and RM tissue extracts. B. HSC-3 monocultures within rinsed myoma (RM) in both normoxic and hypoxic conditions (a, b) show delicate, spindle, caveolin-1-positive cells closely surrounding invading HSC-3 islands. Alpha-smooth muscle actin immunostaining in normoxic and hypoxic conditions (c, d) shows findings similar to caveolin-1 in terms of cell distribution and morphology. HSC-3 monocultures within intact myoma (IM) immunostained with caveolin-1 (e, f) and alpha-smooth muscle (g, h) show findings similar to those of the RM, respectively. CAV1 and alpha-smooth muscle actin positively stain the spindle cells surrounding the invading HSC-3 tumor islands (indicated by arrows). In all sections, the HSC-3 cells are only occasionally CAV1-positive in both RM and IM, and in both normoxia and hypoxia. However, the spindle cells are usually CAV1-positive, showing a high staining intensity.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Caveolin-1 expression in HSC-3 cell myoma monocultures. A. Western blotting: caveolin-1 is detected as complex forms in intact myoma (IM) tissue disc extracts (without added cells), and, to a lesser degree, in the rinsed myoma (RM, without added cells and without soluble factors) disc extracts. Monomeric caveolin-1 is absent from both IM and RM tissue extracts. B. HSC-3 monocultures within rinsed myoma (RM) in both normoxic and hypoxic conditions (a, b) show delicate, spindle, caveolin-1-positive cells closely surrounding invading HSC-3 islands. Alpha-smooth muscle actin immunostaining in normoxic and hypoxic conditions (c, d) shows findings similar to caveolin-1 in terms of cell distribution and morphology. HSC-3 monocultures within intact myoma (IM) immunostained with caveolin-1 (e, f) and alpha-smooth muscle (g, h) show findings similar to those of the RM, respectively. CAV1 and alpha-smooth muscle actin positively stain the spindle cells surrounding the invading HSC-3 tumor islands (indicated by arrows). In all sections, the HSC-3 cells are only occasionally CAV1-positive in both RM and IM, and in both normoxia and hypoxia. However, the spindle cells are usually CAV1-positive, showing a high staining intensity.
Mentions: Although cells within the myoma discs are no longer expected to be viable [19], the microenvironment is rich in soluble factors with necessary functions for facilitating tumor growth and invasion. Depletion of these factors (including CAV1) by rinsing the myoma discs approached a state in which factors released into the myoma assay originated from the cultured HSC-3 cells. Western blotting of either RM or IM discs showed no monomeric 21 kDa form of CAV1 but only complex forms of immunoreactants (Figure 2A). Thus, it can be inferred that CAV1 in the myoma model is expected to originate from the cell lines cultured in this assay.Figure 2

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