A Novel, Tumor-Induced Osteoclastogenesis Pathway Insensitive to Denosumab but Interfered by Cannabidiol

Bone metabolism is strictly regulated, and impaired regulation caused by hormonal imbalances induces systemic bone loss. Local bone loss caused by tumor invasion into bone is suggested to be induced by the generation of cytokines, which affect bone metabolism, by tumor cells. The major cause of systemic and local bone losses is excess bone resorption by osteoclasts, which differentiate from macrophages by receptor activator of nuclear factor kappa-B ligand (RANKL) or tumor necrosis factor-alpha (TNF-alpha). We previously found a novel pathway for tumor-induced osteoclastogenesis targeting osteoclast precursor cells (OPCs). Tumor-induced osteoclastogenesis was resistant to RANKL and TNF-alpha inhibitors. In the present study, we confirmed that exosomes derived from oral squamous cell carcinoma (OSCC) cells induced osteoclasts from OPCs. We also showed that the depletion of exosomes from culture supernatants of OSCC cells partially interfered with osteoclastogenesis, and cannabidiol, an innoxious cannabinoid without psychotropic effects, almost completely suppressed tumor-induced osteoclastogenesis. Osteoclastogenesis and its interference by cannabidiol were independent of the expression of nuclear factor of T cell c1 (NFATc1). These results show that osteoclastogenesis induced by OSCC cells targeting OPCs is a novel osteoclastogenic pathway independent of NFATc1 expression that is partially caused by tumor-derived exosomes and suppressed by cannabidiol

Leukemia inhibitory factor produced by fibroblasts within tumor stroma participates in invasion of oral squamous cell carcinoma

<div><p>The interaction between cancer cells and the cancer stroma plays a crucial role in tumor progression and metastasis in diverse malignancies, including oral cancer. However, the mechanism underlying this interaction remains incompletely elucidated. Here, to investigate the interaction between oral cancer cells and fibroblasts, which are major cellular components of the tumor stroma, we conducted an <i>in vitro</i> study by using human oral squamous cell carcinoma (OSCC) cell lines and normal human dermal fibroblasts (NHDFs). The results of transwell assays revealed that the migration and invasion of 2 OSCC cell lines, HO1-N-1 and HSC3, were markedly stimulated upon coculturing with NHDFs. To investigate the factors that promote tumor invasion, we isolated NHDFs from cocultures prepared with HO1-N-1 cells and performed microarray analysis. Among the various genes that were upregulated, we identified the gene encoding leukemia inhibitory factor (LIF), and we focused on LIF in further analyses. We confirmed that all OSCC-derived conditioned media potently upregulated LIF expression in NHDFs, and the results of our transwell analysis demonstrated that NHDF-induced OSCC migration and invasion were inhibited by LIF-neutralizing antibodies. Furthermore, immunohistochemical analysis of patient samples revealed that in 44 out of 112 OSCC cases, LIF was expressed in the tumor stroma, particularly in cancer-associated fibroblasts (CAFs), and, notably, clinicopathological analyses confirmed that LIF expression in CAFs was significantly correlated with increased depth of tumor invasion. Collectively, our results suggest that OSCC stimulates fibroblasts to produce LIF, which, in turn, participates in cancer-cell invasion. Our finding offers a potential therapeutic strategy targeting the cancer stroma for the treatment of OSCC patients.</p></div

LIF expression in CAFs in OSCC.

<p><b>A:</b> Histology of OSCC invasive front (H&E staining). Scale bar: 100 μm. <b>B:</b> Distribution of LIF-positive fibroblastic cells within the tumor stroma. <b>C:</b> Distribution of α-SMA-positive fibroblastic cells within the tumor stroma. <b>D-F:</b> Double-immunofluorescence staining for LIF (green) and α-SMA (red). Most fibroblasts in the cancer stroma showed colocalization of LIF and α-SMA, but some of the LIF-expressing fibroblasts were negative for α-SMA. Scale bar: 100 μm. Dotted lines in A-C and E-G indicate the interface of cancer nests and the stroma. <b>G:</b> CM derived from 4 OSCC cell lines were added to NHDFs, and α-SMA expression levels in NHDFs were assessed using quantitative real-time PCR analysis after culturing for 48 h. <b>H:</b> LIF expression in human OSCC cell lines. LIF expression was confirmed through RT-PCR analysis. A549, a human lung adenocarcinoma cell line, was used as a positive control. <b>I:</b> LIF expression in NHDFs and HSC3 cells in coculture; 5×10⁴ NHDFs and 5×10³ HSC3 cells were cocultured in 24-well plates. The experiments were performed in triplicate. After 72 h, NHDFs and HSC3 cells were separately isolated from the cocultures through positive and negative selection by using the MACS system. LIF expression was investigated using quantitative real-time PCR analysis. Data represent means ± SEM. ***p < 0.0001.</p

Analysis of overall survival associated with LIF expression in CAFs in human OSCC.

<p><b>A:</b> Kaplan-Meier curve for overall survival in relation to the presence of CAFs in 112 human OSCC cases. <b>B:</b> Kaplan-Meier curve for overall survival in relation to the presence of LIF in CAFs in 112 human OSCC cases.</p

OSCC cell lines stimulate LIF expression in NHDFs.

<p>CM derived from 4 OSCC cell lines were separately added to NHDFs, and increased LIF expression was confirmed through quantitative real-time PCR analysis after 48 h of culture. The experiment was performed in triplicate in 24-well plates. Representative graph from 3 independent experiments is shown. Data represent means ± SEM. Multiple comparisons were performed by using one-way ANOVA with Dunnett’s method. **p < 0.01 compared with control NHDFs.</p

Histology of the tumor invasive region in human OSCC.

<p><b>A, B:</b> Representative histological images of human OSCC cases. Desmoplastic reaction was observed around invasive cancer nests. H&E staining. Scale bar: 100 μm (A) and 200 μm (B).</p

Effect of anti-LIF antibody on migration and invasion of HSC3 cells.

<p>Transwell migration (<b>A</b>) and invasion (<b>B</b>) assays performed using cell-culture inserts. In the upper chamber, 2×10⁴ HSC3 cells were seeded, and 1×10⁵ NHDFs were seeded into the lower well. In each experiment, cells were treated with 0.5 μg/mL of an anti-LIF neutralizing antibody. IgG was used as a control. After 48 h, the migrated or invaded HSC3 cells on the lower surface of the upper chamber were stained and quantified. Representative images and graphs of 3 independent experiments are shown. Multiple comparisons were performed by using one-way ANOVA with Tukey’s method. *p < 0.05, **p < 0.01 compared with control NHDFs. Data represent means ± SEM.</p

Fibroblasts promote migration and invasion of OSCC cells.

<p>Transwell migration (<b>A</b>) and invasion (<b>B</b>) assays performed using cell-culture inserts. In the upper chamber, 2×10⁴ OSCC cells (HSC3, HO1-N-1) were seeded, and 1×10⁵ NHDFs were seeded into the lower well. After 48 h, the OSCC cells that had migrated or invaded into the lower surface of the upper chamber were stained and quantified. Representative images and graphs of 3 independent experiments are shown. ***p < 0.001 compared with control. Data represent means ± SEM.</p