Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma

• Published in: British journal of cancer 2017-09, Vol.117 (6), p.867-875
• Main Authors: Elmusrati, A A, Pilborough, A E, Khurram, S A, Lambert, D W
• Format: Article
• Language: English
• Subjects: 692/4028/67/1059/602; 692/4028/67/322; 692/4028/67/327; 692/699/67/1665; Acid phosphatase; Acid phosphatase (tartrate-resistant); Acid resistance; Actins - analysis; Biomedical and Life Sciences; Biomedicine; Bone and Bones - chemistry; Bone and Bones - drug effects; Bone and Bones - pathology; Bone cancer; Bone Neoplasms - chemistry; Bone Neoplasms - pathology; Bone resorption; Bone turnover; Cancer; Cancer Research; Cancer-Associated Fibroblasts - chemistry; Cancer-Associated Fibroblasts - physiology; Carcinoma, Squamous Cell - chemistry; Carcinoma, Squamous Cell - pathology; Cell culture; Cell Differentiation; Cell Line, Tumor; Conditioning; Culture media; Drug Resistance; Enzyme-linked immunosorbent assay; Epidemiology; Fibroblasts; Gene expression; Humans; Immunohistochemistry; Invasiveness; Macrophages; Malignancy; Mitochondrial Proteins - analysis; Molecular Diagnostics; Molecular Medicine; Molecular modelling; Mouth Neoplasms - chemistry; Mouth Neoplasms - pathology; Neoplasm Invasiveness; Neoplasm Proteins - analysis; Oncology; Oral cancer; Oral carcinoma; Oral squamous cell carcinoma; Osteoblasts; Osteoclastogenesis; Osteogenesis; Osteoprotegerin; Osteoprotegerin - analysis; Proteins; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B - analysis; Reviews; Ribosomal Proteins - analysis; RNA, Messenger - metabolism; Secretion; Squamous cell carcinoma; Stroma; TRANCE protein; Transforming Growth Factor beta1 - pharmacology; Transforming growth factor-b1; Tumors; Western blotting
• ISSN: 0007-0920; 1532-1827
• DOI: 10.1038/bjc.2017.239

Background: The molecular mechanisms involved in the invasion of bone by oral squamous cell carcinomas (OSCC) are poorly understood, and little is known about the role of cancer-associated fibroblasts (CAF), the presence of which confers a poor prognosis. Methods: Clinicopathological data from 277 OSCC cases involving bone resections were reviewed, and 32 cases thoroughly analysed histologically. Immunohistochemistry was used to examine α SMA, RANKL and OPG. Western blotting and qPCR were used to assess myofibroblast (CAF-like) differentiation, RANKL and OPG expression in vitro , and RANKL secretion was analysed by ELISA. Osteoclastogenesis was examined using TRAP staining, multinucleation and pit forming assays. Results: Fibrous stroma intervened between tumour and bone in the majority of cases, with no direct contact between cancer cells and bone. RANKL and OPG, two proteins key to regulating bone resorption, were expressed in tumour cells as well as fibrous stroma adjacent to bone and α SMA-positive myofibroblastic CAF were consistently seen infiltrating into bone ahead of tumour cells. Human primary osteoblasts cultured with conditioned media from human OSCC-derived cells and human primary CAF showed a significant increase in RANKL and a decline in OPG mRNA expression. RANKL secretion was significantly increased in primary oral fibroblasts induced to differentiate into a CAF-like phenotype by transforming growth factor- β 1 (TGF- β 1) treatment and in primary CAF. Indirect co-culture of murine macrophages with conditioned media from CAF (experimentally derived and isolated from OSCCs) resulted in a marked increase in osteoclastogenesis (in excess of that provoked by cancer cells) determined by tartrate-resistant acid phosphatase activity, multinucleation and resorption pit formation. Conclusions: This study is the first to describe a functional role for CAFs in bone invasion and turnover, identifying a novel potential therapeutic target and diagnostic indicator in this difficult to treat bone invasive malignancy.