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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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Related in: MedlinePlus

Caveolin-1 in monolayer HSC-3 extracts (A) and in exosomes isolated from their media (B-D). A. Caveolin-1 protein are seen as strong monomeric bands (~21 kDa) (and in complex forms, ~50 kDa – 60 kDa) in whole HSC-3 cell extracts cultured in monolayers under normoxic (NOR) conditions. Hypoxic (HYP) oxidative conditions and hypoxia mimicking CoCl2 media yield similar results. B. Exosomes isolated from the media of HSC-3 normoxic monolayer submitted to Western blotting for caveolin-1 show strong bands in the 20–23 kDa form (and in complex forms, ~50 kDa – 60 kDa). C. Results of ELISA tests performed on the exosomes isolated from HSC-3 monolayer media reveal caveolin-1 expression. The results are presented as the mean amount (±SD) of each protein of experiments with and without APMA induction. D. ELISA performed on HSC-3 exosomes shows the expression of the exosomal markers, CD63, CD9 and CD81. The results are presented as the mean amount (±SD) of each protein, with and without APMA induction. E. Immuno-electron microscopy that was performed on the monolayer media-isolated exosomes features round-shaped, membrane-bound nanoparticles that contain dots positive for both caveolin-1 and CD63.
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Fig5: Caveolin-1 in monolayer HSC-3 extracts (A) and in exosomes isolated from their media (B-D). A. Caveolin-1 protein are seen as strong monomeric bands (~21 kDa) (and in complex forms, ~50 kDa – 60 kDa) in whole HSC-3 cell extracts cultured in monolayers under normoxic (NOR) conditions. Hypoxic (HYP) oxidative conditions and hypoxia mimicking CoCl2 media yield similar results. B. Exosomes isolated from the media of HSC-3 normoxic monolayer submitted to Western blotting for caveolin-1 show strong bands in the 20–23 kDa form (and in complex forms, ~50 kDa – 60 kDa). C. Results of ELISA tests performed on the exosomes isolated from HSC-3 monolayer media reveal caveolin-1 expression. The results are presented as the mean amount (±SD) of each protein of experiments with and without APMA induction. D. ELISA performed on HSC-3 exosomes shows the expression of the exosomal markers, CD63, CD9 and CD81. The results are presented as the mean amount (±SD) of each protein, with and without APMA induction. E. Immuno-electron microscopy that was performed on the monolayer media-isolated exosomes features round-shaped, membrane-bound nanoparticles that contain dots positive for both caveolin-1 and CD63.

Mentions: First, we investigated CAV1 expression in HSC-3 cell extracts in normoxia as well as in hypoxic (1%O2) and hypoxia-mimicking (CoCl2) conditions, similarly to what had been investigated in the myoma model. Western blotting for CAV1 detected a corresponding 21 kDa band in normoxia and bands of a similar width in the hypoxic conditions. This highlights the ability of HSC-3 cells to express CAV1 and demonstrates that this ability was not influenced by hypoxia (Figure 5A). In addition, Western blotting for CAV1 performed on the media of the monolayer cells was negative (data not shown).Figure 5


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 in monolayer HSC-3 extracts (A) and in exosomes isolated from their media (B-D). A. Caveolin-1 protein are seen as strong monomeric bands (~21 kDa) (and in complex forms, ~50 kDa – 60 kDa) in whole HSC-3 cell extracts cultured in monolayers under normoxic (NOR) conditions. Hypoxic (HYP) oxidative conditions and hypoxia mimicking CoCl2 media yield similar results. B. Exosomes isolated from the media of HSC-3 normoxic monolayer submitted to Western blotting for caveolin-1 show strong bands in the 20–23 kDa form (and in complex forms, ~50 kDa – 60 kDa). C. Results of ELISA tests performed on the exosomes isolated from HSC-3 monolayer media reveal caveolin-1 expression. The results are presented as the mean amount (±SD) of each protein of experiments with and without APMA induction. D. ELISA performed on HSC-3 exosomes shows the expression of the exosomal markers, CD63, CD9 and CD81. The results are presented as the mean amount (±SD) of each protein, with and without APMA induction. E. Immuno-electron microscopy that was performed on the monolayer media-isolated exosomes features round-shaped, membrane-bound nanoparticles that contain dots positive for both caveolin-1 and CD63.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: Caveolin-1 in monolayer HSC-3 extracts (A) and in exosomes isolated from their media (B-D). A. Caveolin-1 protein are seen as strong monomeric bands (~21 kDa) (and in complex forms, ~50 kDa – 60 kDa) in whole HSC-3 cell extracts cultured in monolayers under normoxic (NOR) conditions. Hypoxic (HYP) oxidative conditions and hypoxia mimicking CoCl2 media yield similar results. B. Exosomes isolated from the media of HSC-3 normoxic monolayer submitted to Western blotting for caveolin-1 show strong bands in the 20–23 kDa form (and in complex forms, ~50 kDa – 60 kDa). C. Results of ELISA tests performed on the exosomes isolated from HSC-3 monolayer media reveal caveolin-1 expression. The results are presented as the mean amount (±SD) of each protein of experiments with and without APMA induction. D. ELISA performed on HSC-3 exosomes shows the expression of the exosomal markers, CD63, CD9 and CD81. The results are presented as the mean amount (±SD) of each protein, with and without APMA induction. E. Immuno-electron microscopy that was performed on the monolayer media-isolated exosomes features round-shaped, membrane-bound nanoparticles that contain dots positive for both caveolin-1 and CD63.
Mentions: First, we investigated CAV1 expression in HSC-3 cell extracts in normoxia as well as in hypoxic (1%O2) and hypoxia-mimicking (CoCl2) conditions, similarly to what had been investigated in the myoma model. Western blotting for CAV1 detected a corresponding 21 kDa band in normoxia and bands of a similar width in the hypoxic conditions. This highlights the ability of HSC-3 cells to express CAV1 and demonstrates that this ability was not influenced by hypoxia (Figure 5A). In addition, Western blotting for CAV1 performed on the media of the monolayer cells was negative (data not shown).Figure 5

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