Osteoclasts Are Active in Bone Forming Metastases of Prostate Cancer Patients

BACKGROUND: Bone forming metastases are a common and disabling consequence of prostate cancer (CaP). The potential role of osteoclast activity in CaP bone metastases is not completely explained. In this study, we investigated ex vivo whether the osteolytic activity is present and how it is ruled in CaP patients with bone forming metastases. METHODOLOGY: Forty-six patients affected by newly diagnosed CaP and healthy controls were enrolled. At diagnosis, 37 patients had a primary tumour only, while 9 had primary tumour and concomitant bone forming metastases. In all patients there was no evidence of metastasis to other non-bone sites. For all patients and controls we collected blood and urinary samples. We evaluated patients' bone homeostasis; we made peripheral blood mononuclear cell (PBMC) cultures to detect in vitro osteoclastogenesis; we dosed serum expression of molecules involved in cancer induced osteoclatogenesis, such as RANKL, OPG, TNF-alpha, DKK-1 and IL-7. By Real-Time PCR, we quantified DKK-1 and IL-7 gene expression on micro-dissected tumour and healthy tissue sections. PRINCIPAL FINDINGS: CaP bone metastatic patients showed bone metabolism disruption with increased bone resorption and formation compared to non-bone metastatic patients and healthy controls. The CaP PBMC cultures showed an enhanced osteoclastogenesis in bone metastatic patients, due to an increase of RANKL/OPG ratio. We detected increased DKK-1 serum levels and tissue gene expression in patients compared to controls. IL-7 resulted high in patients' sera, but its tissue gene expression was comparable in patients and controls. CONCLUSIONS: We demonstrated ex vivo that osteoclastogenesis is an active mechanism in tumour nesting of bone forming metastatic cancer and that serum DKK-1 levels are increased in CaP patients, suggesting to deeply investigate its role as tumour marker

Characteristics of patients and healthy controls.

<p>Bone turnover marker values are shown as mean±SD, the p values were calculated by one way ANOVA and the Bonferroni post-hoc correction. * and ° indicates the values significantly different between patients with/ without bone metastases (* <i>p</i> = 0.001, ° <i>p</i> = 0.000).</p

DKK-1 expression is higher in CaP patients.

<p>DKK-1 levels were dosed in serum patients with/without bone metastases and in healthy controls by ELISA. Bone metastatic (<i>p</i><0.004) and non-bone metastatic patients (<i>p</i><0.01) had significantly higher DKK-1 serum levels compared to healthy controls (A). CaP and healthy tissues were analyzed by Real-Time PCR in order to quantify DKK-1gene expression. The DKK-1 quantization was expressed as DKK-1 on β-Actin (the control gene) plasmid copy number. The histogram showed higher DKK-1 expression levels in CaP than in healthy tissues, <i>p</i><0.001 (B).</p

IL-7 increases OC bone resorbing activity.

<p>PBMC resorption activity with/without IL-7 were showed for bone metastatic patients <b>(line A)</b> and healthy controls <b>(line B)</b>. The basal resorbing activity of OCs derived from patients was significantly higher compared to healthy controls, ^#<i>p</i>≤0,0001 <b>(C)</b>. The percentage of resorption area was significantly increased with 2,5 ng/ml of IL-7, both in patients and in healthy controls, *<i>p</i>≤0,002 <b>(C)</b>. At 15 ng/ml IL-7 did not elicit a significant variation of resorption area. The data represent the means±SE from ten independent experiments.</p

Effect of anti-TNF-α and OPG on IL-7-induced osteoclastogenesis.

<p>The addition of both OPG and anti-TNF-α caused a dose-dependent inhibition of IL-7-induced osteoclastogenesis. The anti-TNF-α antibody caused a strong osteoclastogenesis inhibition, about 90% <b>(A)</b> while OPG caused a 50% inhibition <b>(B)</b>.</p

Effect of IL-7 on osteoclastogenesis <i>in vitro.</i>

<p>TRAP⁺ multinucleated cells were identified as OCs at the end of culture in both bone metastatic patients <b>(line A)</b> and healthy controls <b>(line B)</b>. PBMC cultures were plated and stimulated with IL-7 (0,5–1–2,5–10–15 ng/ml). The osteoclastogenesis significantly increased at 2,5 ng/ml of IL-7 compared to the unstimulated condition <b>(C)</b>. The anti-IL-7 antibody added in cell culture determined a dose-dependent inhibition of spontaneous osteoclastogenesis in bone metastatic patients <b>(D)</b>. Multinucleated/TRAP positive cells, derived both from culture with and without IL-7, expressed also α_Vβ₃<b>(E)</b>.</p

IL-7 levels in cell culture medium and serum.

<p>IL-7 levels in cancer patients with/without bone metastases and in healthy controls were analysed by ELISA. At day 5, supernatants of patients' PBMC cultures and monocyte plus T and B cell co-colture had IL-7 levels higher than healthy controls (A). Bone metastatic patients had significantly higher serum levels of IL-7 compared to patients without bone metastasis and healthy controls (B). Samples were assayed in duplicate and data were expressed as mean values.</p

IL-7 stimulates osteoclastogenesis in co-coltures of monocytes and T or B cells.

<p>The different subpopulations were isolated from PBMCs and co-coltures of monocytes and T or B cells (M+T; M+B) were made. By adding 2,5 ng/ml of IL-7 in co-coltures we observed an increase in osteoclastogenesis, which was statistically significant for T cells <b>(A)</b> and not for B cells <b>(B)</b>. At 15 ng/ml of IL-7, there were not significant effects in both the co-coltures.</p