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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 monocultures with two types of triple immunohistochemical panels. Caveolin-1 (CAV1), Twist and cytokeratin (CK) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or in hypoxia chamber with 1% O2)] (a-f) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with cytokeratin and, to a lesser extent, with CAV1 (arrows). In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are also CAV1-positive. CK and alpha-smooth muscle actin (αSMA) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or 1%O2)] (g-l) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with CK. In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are αSMA-positive.
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Fig3: HSC-3 monocultures with two types of triple immunohistochemical panels. Caveolin-1 (CAV1), Twist and cytokeratin (CK) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or in hypoxia chamber with 1% O2)] (a-f) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with cytokeratin and, to a lesser extent, with CAV1 (arrows). In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are also CAV1-positive. CK and alpha-smooth muscle actin (αSMA) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or 1%O2)] (g-l) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with CK. In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are αSMA-positive.

Mentions: Since in the previously described experiment (i.e., IM and RM HSC-3 monocultures in normoxic and hypoxic conditions) the CAV1-positive spindle-shaped cells were found in an intimate relation to the HSC-3 tumor clusters, we raised the possibility that the origin of these cells could be from HSC-3 cells that had undergone a process of epithelial-mesenchymal transition (EMT). Therefore, serial sections from that experiment were triple immunostained with CAV1, pan-cytokeratin and Twist, an EMT marker. We found that invading cytokeratin-positive HSC-3 cells in normoxic RM sections had a nuclear expression of Twist, and that a number of cells also showed cytoplasmic expression of CAV1 (Figure 3). The delicate spindle-shaped cells that were concentrically arranged in the closest periphery of the HSC-3 cells were CAV1-positive. This configuration of the spindle cells was identified exclusively at the periphery of invading HSC-3 cells and not in areas of myoma beyond this vicinity or in areas of myoma devoid of HSC-3 cells. These findings were similar to those of hypoxic RM sections, thus once more supporting the conclusions that a myoma constitutes a natural hypoxic environment and that factors secreted from the HSC-3 cells can have autocrine and/or paracrine effects. These findings are reflected by the evolvement of the spindle cells at the periphery of the HSC-3 tumor islands. Normoxic and hypoxic IM sections showed a similar set of results.Figure 3


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 monocultures with two types of triple immunohistochemical panels. Caveolin-1 (CAV1), Twist and cytokeratin (CK) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or in hypoxia chamber with 1% O2)] (a-f) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with cytokeratin and, to a lesser extent, with CAV1 (arrows). In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are also CAV1-positive. CK and alpha-smooth muscle actin (αSMA) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or 1%O2)] (g-l) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with CK. In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are αSMA-positive.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: HSC-3 monocultures with two types of triple immunohistochemical panels. Caveolin-1 (CAV1), Twist and cytokeratin (CK) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or in hypoxia chamber with 1% O2)] (a-f) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with cytokeratin and, to a lesser extent, with CAV1 (arrows). In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are also CAV1-positive. CK and alpha-smooth muscle actin (αSMA) in rinsed and intact myoma in normoxia and hypoxia [cobalt chloride (CoCl2) or 1%O2)] (g-l) show similar patterns of expression in all settings: positive nuclear staining of the invading HSC-3 islands with Twist (most nuclei) and diffuse cytoplasmic staining with CK. In addition, delicate, spindle-shaped cells that closely surround these invading tumor islands in a concentric pattern are αSMA-positive.
Mentions: Since in the previously described experiment (i.e., IM and RM HSC-3 monocultures in normoxic and hypoxic conditions) the CAV1-positive spindle-shaped cells were found in an intimate relation to the HSC-3 tumor clusters, we raised the possibility that the origin of these cells could be from HSC-3 cells that had undergone a process of epithelial-mesenchymal transition (EMT). Therefore, serial sections from that experiment were triple immunostained with CAV1, pan-cytokeratin and Twist, an EMT marker. We found that invading cytokeratin-positive HSC-3 cells in normoxic RM sections had a nuclear expression of Twist, and that a number of cells also showed cytoplasmic expression of CAV1 (Figure 3). The delicate spindle-shaped cells that were concentrically arranged in the closest periphery of the HSC-3 cells were CAV1-positive. This configuration of the spindle cells was identified exclusively at the periphery of invading HSC-3 cells and not in areas of myoma beyond this vicinity or in areas of myoma devoid of HSC-3 cells. These findings were similar to those of hypoxic RM sections, thus once more supporting the conclusions that a myoma constitutes a natural hypoxic environment and that factors secreted from the HSC-3 cells can have autocrine and/or paracrine effects. These findings are reflected by the evolvement of the spindle cells at the periphery of the HSC-3 tumor islands. Normoxic and hypoxic IM sections showed a similar set of results.Figure 3

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