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Mechanisms of spontaneous osteoclastogenesis in cancer with bone involvement

Ilaria Roato^*,,1, Maria Grano, Giacomina Brunetti, Silvia Colucci, Antonio Mussa, Oscar Bertetto and Riccardo Ferracini

^* CeRMS (Center for Experimental Research and Medical Studies) University and A.S.O. San Giovanni Battista, Turin, Italy;
Department of Human Anatomy and Histology, University of Bari, Italy;
Department of Medical Oncology; University and A.S.O. San Giovanni Battista, Turin, Italy; and
Department of Orthopaedics, A.S.O. San Giovanni Battista, Turin, Italy

¹Correspondence: Laboratory of CeRMS, A.S.O. San Giovanni Battista, Via Santena 5, Torino 10126, Italy. E-mail: roato78{at}libero.it

SPECIFIC AIMS

The major aim of our study concerns the comprehension of the mechanisms of bone osteolytic metastases in solid tumors. We investigated whether committed osteoclast precursors (OCPs) were activated in peripheral blood of metastatic patients with bone involvement. We collected blood samples from patients with different solid tumors with or without metastatic bone lesions in order to study osteoclastogenesis in peripheral blood mononuclear cell (PBMC) cultures.

PRINCIPAL FINDINGS

1. Spontaneous osteoclastogenesis in unstimulated PBMCs from patients affected by solid tumors
We investigated the osteoclastogenic potential of PBMCs from patients affected by solid tumors as well as from controls cultured with or without addition of M-CSF, RANKL, and TNF-. Numerous large TRAP⁺ osteoclasts (OCs) were identified in unstimulated PBMC cultures from cancer patients with osteolysis (Fig. 1 D), whereas smaller and fewer OCs appeared in PBMC cultures from controls (Fig. 1B ) and cancer patients without osteolysis (data not shown). The addition of M-CSF and RANKL to PBMC cultures from patients with osteolysis did not significantly change the OC number (Fig. 1C ), while these cytokines were essential to trigger and sustain PBMCs’ osteoclastogenesis from controls (Fig. 1A ) and from patients without osteolysis. Addition of exogenous M-CSF and TNF- to PBMC cultures of patients without osteolysis significantly increased the number of OCs (Fig. 1F ) compared with controls (Fig. 1E ) and to OCs obtained by adding M-CSF/RANKL to the same cultures (data not shown). Since increasing doses of RANKL did not further enhance osteoclastogenesis, we assume that this mechanism is RANKL-independent. To confirm RANKL-independence of the described activation, we demonstrated that the addition of OPG to the PBMC culture was unable to modify spontaneous osteoclastogenesis.

View larger version (75K): [in this window] [in a new window]   Figure 1. OCs generated from human cancer patients PBMCs. OCs were obtained from PBMCs of cancer patients with bone metastases (C, D), without bone metastases (F) and controls (A, B, E). Numerous and large-sized OCs (arrows) developed in the unstimulated cultures from osteolytic cancer patients (D) whereas rare and small-sized OCs were observed in the cultures from controls (B) and nonosteolytic patients. No significant increase in OC formation was observed in PBMCs from cancer patients with bone lesions after stimulation with exogenous M-CSF and RANKL (C). Addition of M-CSF/TNF- induced OCs formation in PBMC cultures of patients without bony involvement (F). M-CSF, RANKL, and TNF- were essential to trigger the OC formation in controls (A, E). Multinucleated (>3 nuclei/cell) and TRAP+ cells were identified as OCs. Arrows point to the OCs (x200).

In osteolytic patients’ PBMC cultures the presence of different concentrations of a neutralizing anti-TNF- antibody inhibited spontaneous osteoclastogenesis in a dose-dependent way. We also tested the addition of TNF- to PBMC cultures of patients and healthy controls and we observed formation of OCs. These results implicate TNF- as the major cytokine involved in spontaneous osteoclastogenesis from PBMCs of patients with bone metastases.

2. The number of circulating osteoclast precursors is higher in cancer patients
To determine whether the increased number of OCs was derived from proliferation of circulating OCPs, we analyzed the presence of surface markers of mononuclear OCPs by flow cytometry. Freshly isolated PBMCs from cancer patients with lytic lesions and controls were stained for CD11b, CD14, and CD51/CD61. These OCP markers were expressed, and the percentage of PBMCs expressing CD11b and CD14 was greater in patients bearing lytic lesions than in controls(P<0.007).

3. Bone-resorbing activity of osteoclasts
To assess bone-resorbing activity, OCs generated from PBMCs of cancer patients with lytic lesions and controls were cultured for 21 days on dentine slices. In cultures of PBMCs, incubated with or without M-CSF and RANKL, resorption pits formation was evident. The bone surface area eroded by OCs from cancer patients was higher than that eroded by controls, (P<0.008). These data demonstrate a functional OCs phenotype: in unstimulated cancer patients PBMCs are capable of enhanced bone-resorbing activity.

4. T cells mediate osteoclastogenesis
To assess the T cells’ potential regulatory role in solid tumor osteoclastogenesis, we established unstimulated cultures of T cell depleted PBMCs from cancer patients with osteolysis that resulted in development of few small-sized OCs. In contrast, the addition of M-CSF and RANKL to these cultures induced the formation of numerous large TRAP⁺ OCs, similar to those observed in unfractionated and unstimulated PBMCs from cancer patients.

5. Gene expression in T lymphocytes and in osteoclasts derived from unstimulated PBMCs
To study the mechanism used by T cells to support OC formation, we analyzed the expression of osteoclastogenic factors possibly involved.

In osteolytic patients, fresh T cells purified from PBMCs overexpressed TNF-, while in samples derived from cancer patients without osteolysis and from the controls a very low expression of TNF- was observed. The same results were obtained in OCs from unstimulated PBMCs of cancer patient with osteolysis, without osteolysis, and from the controls, indicating the OCs ability to stimulate themselves by TNF- in cancer patients. T cells and OCs did not express RANKL mRNA.

CONCLUSIONS AND SIGNIFICANCE

Published reports have demonstrated that multiple myeloma patients show production of cytokines and pro-osteoclastogenic factors that determine differentiation of circulating OCPs, and subsequently their activation into mature OCs.

We have studied patients affected by different types of solid tumors bearing osteolytic lesions: lung, breast, colon, melanoma, kidney, and prostate cancers. We needed data to identify possible similarities or differences in osteoclastogenesis among different histologies and locations. We did not find differences in spontaneous osteoclastogenesis in different tumors, subtypes, number, and location of the metastases. Prostatic patients were selected for osteolytic or so-called "mixed type" (osteolytic and osteoblastic) lesions.

The current literature reports that osteoclastogenesis from PBMCs occurs in vitro and depends on the stimulation with several osteoclastogenesis factors such as M-CSF, RANKL, and TNF-. In this study, we describe a spontaneous osteoclastogenesis in patients affected by solid tumors with metastatic osteolysis. OCs obtained from these patients are fully differentiated, mature, and are active in resorption of bone matrix. These findings suggest that osteoclastogenesis in metastatic bone lesions not only depend on stimulation by factors present in the microenvironment of metastatic sites, but can also be induced by a general and ubiquitous activation of OCPs in blood. This spontaneous osteoclastogenesis seems to correlate directly with the higher number of circulating OCPs (expressing CD14, CD11b, and CD51/CD61) in metastatic patients compared with controls.

Recent literature has reported a regulatory role of bone turnover and OC activation played by T cells in physiological and pathological conditions. Thus, we focused our attention on T cells as possible activators of osteoclastogenesis in vitro. We have demonstrated that in T cell-depleted PBMC cultures derived from cancer patients, the spontaneous osteoclastogenesis observed in unfractionated PBMC cultures is totally reverted in absence of autologous lymphocytes. T cell-depleted PBMCs cultured alone are totally dependent on exogenous factors for in vitro osteoclastogenesis; we therefore have supposed that T cells first receive a priming by the tumor microenvironment, then release osteoclastogenic factors and subsequently continue their activator role ex vivo. We investigated the profile expression of activation and memory markers (CD69, CD25, and CD44) of T cells. Our results on the characterization of the T cell population agree with the literature and show an increase in the percentage of T cells CD25 and CD44-positive.

Since we found that increasing doses of RANKL in culture do not up-regulate osteoclastogenesis and mRNA for TNF- has expressed by lymphocytes and OCs while RANKL mRNA was not present, we assumed that osteoclastogenesis could depend on TNF- released in culture. A pivotal role for TNF- in promoting osteoclastogenesis was confirmed by the observation that addition of an anti-TNF- neutralizing antibody to PBMC cultures determined a dose-dependent osteoclastogenesis inhibition.

The described experiments based on the patient PBMC cultures show osteoclastogenesis when osteolytic bone metastases are present. We observed the absence of spontaneous osteoclastogenesis in controls or in cancer patients without bony localizations. Osteoclastogenesis correlates directly with the clinical course of the disease. These observations suggest that this in vitro assay could be used for diagnostic purposes. We believe that osteoclastogenesis is concomitant to the activation of osteolysis. Activation of osteoclastic precursors via T cells depends on circulating tumor cells or on factors released from the tumor site. Whatever the mechanism of PBMC commitment, this in vitro assay could be used prognostically in cancer patients with a high risk of osteolytic metastases and in the follow-up of these patients, especially after a curative treatment.

View larger version (48K): [in this window] [in a new window]   Figure 2. Osteolytic metastasis is a multistep process. 1) Primary malignant cells proliferate and invade the blood stream, where they interact with T cells and monocytes and/or release activating factors; 2) T cells are activated and release TNF-, which promotes monocyte differentiation to osteoclast precursors (OCPs); 3) tumor cells and OCPs enter in bone and the bone microenvironment supports growth of metastases and maturation of bone resorbing osteoclasts.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1823fje;

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