Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation.

BACKGROUND: Skeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation. METHODS: To dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl-/-) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs). RESULTS: We found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl-/- mice as compared to wild-type. Rankl-/- mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl-/- SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures. CONCLUSIONS: We provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

Dose-finding/phase II trial: bevacizumab, immunotherapy, and chemotherapy (BIC) in metastatic renal cell cancer (mRCC). Antitumor effects and variations of circulating T regulatory cells (Treg).

The aim of this study was to explore the efficacy and toxicities of a combined regimen of bevacizumab plus immunotherapy and chemotherapy (BIC) and the circulating T regulatory cells (Treg) in metastatic renal cell cancer (mRCC). Nephrectomized mRCC patients were enrolled into a multicenter single-arm dose-finding study with five escalated dose levels of chemotherapy with intravenous gemcitabine and 5-fluorouracil associated with fixed intravenous doses of bevacizumab, subcutaneous low doses of interleukin-2, and interferon-α-2a. An expanded cohort (phase II study) was treated at the recommended dose for additional safety and efficacy information according to minimax Simon two-stage design. Blood samples for Treg were collected and evaluated by fluorescence-activated cell sorting (FACS) analysis on cycle 1. Fifty-one patients were entered to receive one of five dose levels. Median age was 58 years (male 67 %, pretreated 49 %): 15 patients were low risk according to Memorial Sloan-Kettering Cancer Center (MSKCC) criteria, while 27 and nine were respectively intermediate- and high-risk patients. More frequent grade 3 and 4 toxicities included nonfebrile neutropenia, thrombocytopenia, and fever. Among patients evaluable for response (49), 29.5 % had partial response and 37 % stable disease. Overall median time to progression and median overall survival were 8.8 and 22.67 months, respectively. We observed a rapid increase in the percentage of Treg after immunotherapy and a reduction after bevacizumab only in patient who obtained a partial response or stable disease. The BIC was feasible, well tolerated, and shown interesting activity. Further studies are needed to explore if Treg could have a role in clinical response in mRCC treated with bevacizumab.