Hedgehog pathway activation in oral squamous cell carcinoma: cancer-associated fibroblasts exhibit nuclear GLI-1 localiz
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ORIGINAL PAPER
Hedgehog pathway activation in oral squamous cell carcinoma: cancer‑associated fibroblasts exhibit nuclear GLI‑1 localization Vanessa Sousa Nazare Guimaraes1,2 · Manuela Torres Andion Vidal1,2 · Ludmila de Faro Valverde1,2 · Marbele Guimarães de Oliveira1,2 · Leonardo de Oliveira Siquara da Rocha1,2 · Paulo Lucas Cerqueira Coelho1 · Fernando Augusto Soares3,4 · Bruno Solano de Freitas Souza1 · Daniel Pereira Bezerra1 · Ricardo D. Coletta5 · Thiago Almeida Pereira6 · Jean Nunes dos Santos2 · Clarissa Araújo Gurgel Rocha1,2,7 Received: 29 April 2020 / Accepted: 22 September 2020 © Springer Nature B.V. 2020
Abstract The purpose of this study was to evaluate the expression of Hedgehog (HH) signaling molecules (SHH and GLI-1) by cancerassociated fibroblasts (CAF) in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to detect molecular HH signaling and CAF-related protein expression, including α-SMA and S100A4, in 70 samples of human OSCC. The colocalization of α-SMA and S100A4 with SHH was also evaluated by double-staining. In vitro study was performed using primary normal oral fibroblast (NOF) and CAF through immunofluorescence and Western Blot for CAF-proteins, SHH, and GLI-1. Forty-five cases (64.28%) were positive for α-SMA exclusively in tumor stroma, and S100A4 was identified in the cytoplasm of CAFs in 94.28% (n = 66) of the cases. With respect to stromal cells, 64 (91.43%) OSCC cases were positive for SHH, and 31 were positive for GLI-1 (44.29%); positive correlations were found between SHH and α-SMA (p 0.5 was adopted to indicate a strong positive association, in accordance with Davis (1971).
Results Heterogeneity of CAF population in OSCC The α-SMA protein was diffusely distributed in 45 of the OSCC cases (64.28%), and identified exclusively in the membranes and cytoplasm of those stromal cells morphologically similar to fibroblasts located adjacent to tumor islets, with a predominant score of 3 + (n = 24, 53.33%). Positivity for the S100A4 protein was also seen in fibroblasts (3 + score: n = 41, 62.13%), and focal positivity was observed in endothelial, inflammatory and malignant cells (1 + score: n = 34, 66.67%). Remaining scores are described in Table 2. While all TFMs were negative for α-SMA, immunostaining for S100A4 was observed in 90% (n = 9) of the stromal cells in the TFMs included in the study (3 + score: n = 4; 44.44%). Figure 2 illustrates the immunostaining patterns found for α-SMA and S100A4 in OSCC tumor stroma
Journal of Molecular Histology Table 2 Immunostaining score for tumors cells and stromal cells
Immunohistochemical expression of proteins Tumor cells 0
α-SMA S100A4 SHH GLI1
Stromal cells 1 +
2 +
3 +
0
1 +
n
%
n
%
n
%
n
%
n
70 19 6 15
100 27.14 8.57 21.42
0 34 8 15
0 48.58 11.43 21.42
0 13 6 12
0 18.58 8.57 17.16
0 4 50 28
0 5.7 71.43 40
25 35.72 13 4 5.72 8 31 44.28 23 39 55.71 23
Fig. 2 Immunomarkers expressed by CAFs in OSCC and TFM. All analyses were performed in identical fields. Diffuse and abundant immunostainin
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