Osteoclasts are active in bone forming metastases of prostate cancer patients

Abstract

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.

Figure 1. Analysis of osteoclastogenesis from CaP patients' PBMCs.

TRAP positive multinucleated cells were identified as OCs and counted, in both patients and healthy controls cultures, (A). The OC number in bone metastatic patients was significantly higher than in non-bone metastatic patients, p<0.004 and in healthy controls, p<0.001 (B).

Figure 2. IL-7 expression by CaP.

IL-7 serum levels in patients with/without bone metastases and in healthy controls were measured by ELISA. Bone metastatic (p<0.01) and non-bone metastatic patients (p<0.03) had significantly higher IL-7 serum levels compared to healthy controls (A). CaP and healthy tissues were analyzed by Real-Time PCR in order to quantify IL-7 gene expression. The IL-7 quantization was expressed as IL-7 on β-Actin (the control gene) plasmid copy number. The histogram showed comparable IL-7 expression levels in CaP and healthy tissues.

Figure 3. DKK-1 expression is higher in CaP patients.

DKK-1 levels were dosed in serum patients with/without bone metastases and in healthy controls by ELISA. Bone metastatic (p<0.004) and non-bone metastatic patients (p<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, p<0.001 (B).