Tumor Biology

, Volume 37, Issue 7, pp 9233–9241 | Cite as

Macrophages and endothelial cells orchestrate tumor-associated angiogenesis in oral cancer via hedgehog pathway activation

  • Ludmila de Faro Valverde
  • Thiago de Almeida Pereira
  • Rosane Borges Dias
  • Vanessa Sousa Nazaré Guimarães
  • Eduardo Antônio Gonçalves Ramos
  • Jean Nunes Santos
  • Clarissa Araújo Gurgel Rocha
Original Article


The present study aimed to evaluate the role of Hedgehog (Hh) molecule expression in association with the clinical aspects of oral squamous cell carcinoma (OSCC), as well as angiogenesis and CD163+ macrophages. Twenty-eight cases of OSCC, nine cases of tumor-free resection margins (TM), and four cases of non-neoplastic oral mucosa (NNM) were submitted to immunohistochemistry to detect proteins Sonic Hedgehog (SHH), Indian Hedgehog (IHH), GLI1, CD163, and CD105. Protein colocalization with respect to SHH/CD163, IHH/CD163, GLI1/CD163, and GLI1/CD105 was assessed by immunohistochemical double staining. In tumor parenchyma, SHH and IHH were present in the cytoplasm of neoplastic cells, while GLI1 was observed in cytoplasm and nucleus. Endothelial cells were found to express SHH, IHH, and GLI1 within CD105+ vessels, and a positive correlation between infiltrating macrophage density (IMD) and microvascular density (MVD) was observed in cases of OSCC and TM. When compared to TM and NNM, the OSCC cases demonstrated higher immunoreactivity for SHH (p = 0.01), IHH (p = 0.39), GLI1 (p = 0.03), IMD (p = 0.0002), and MVD (p = 0.0002). Our results suggest the participation of the Hh pathway in OSCC by way of autocrine and paracrine signaling, in addition to the participation of both SHH and IHH ligands. Endothelial cells were also found to exhibit positivity with respect to Hh pathway components and we surmise that these molecules may play a role in tumor angiogenesis. CD163+ macrophages were also observed to express IHH, a ligand of this pathway, in addition to being associated with tumor neovascularization.


Oral cancer Squamous cell carcinoma Hedgehog proteins Immunohistochemistry 



This study was supported by grants from Foundation for Search of Bahia State—FAPESB, Bahia, Brazil (007/2013), and National Council for Scientific and Technological Development, CNPq, Brazil (446065/2014-5). The authors would like to thank Andris K. Walter for providing English translation and consulting services.

Compliance with ethical standards

Conflicts of interest


Ethics approval and consent to participate

The present research proposal received approval from our host institution’s review board. Informed consent was obtained from all individual participants included in the study.

Supplementary material

13277_2015_4763_Fig7_ESM.jpg (163 kb)
Supplemental Fig. 1

(a) CD163 immunoreactivity (Permanent Red) evident in the membrane and cytoplasm in OSCC cases. (b) IHH immunoreactivity (Vina Green) in the membrane and cytoplasm in OSCC cases. (JPG 163 kb)

13277_2015_4763_MOESM1_ESM.tif (8.1 mb)
High resolution image (TIF 8319 kb)
13277_2015_4763_MOESM2_ESM.doc (78 kb)
Supplemental Table 1 (DOC 78 kb)
13277_2015_4763_MOESM3_ESM.doc (40 kb)
Supplemental Table 2 (DOC 40 kb)


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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Ludmila de Faro Valverde
    • 1
  • Thiago de Almeida Pereira
    • 2
  • Rosane Borges Dias
    • 1
  • Vanessa Sousa Nazaré Guimarães
    • 1
  • Eduardo Antônio Gonçalves Ramos
    • 1
  • Jean Nunes Santos
    • 3
  • Clarissa Araújo Gurgel Rocha
    • 1
    • 3
  1. 1.Laboratory of Pathology and Molecular Biology, Oswaldo Cruz FoundationGonçalo Moniz Research CenterSalvadorBrazil
  2. 2.Laboratory of Experimental PathologyOswaldo Cruz FoundationSalvadorBrazil
  3. 3.Laboratory of Oral Surgical PathologySchool of Dentistry of the Federal University of BahiaSalvadorBrazil

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