The Intrinsic and Extrinsic Implications of RANKL/RANK Signaling in Osteosarcoma: From Tumor Initiation to Lung Metastases

Background: Osteosarcoma is the most frequent form of malignant pediatric bone tumor. Despite the current therapeutic arsenal, patient life-expectancy remains low if metastases are detected at the time of diagnosis, justifying research into better knowledge at all stages of osteosarcoma ontogenesis and identification of new therapeutic targets. Receptor Activator of Nuclear factor κB (RANK)expression has been reported in osteosarcoma cells, raising the question of Receptor Activator of Nuclear factor κB Ligand (RANKL)/RANK signaling implications in these tumor cells (intrinsic), in addition to previously reported implications through osteoclast activation in the tumor microenvironment (extrinsic). Methods: Based on in vitro and in vivo experimentations using human and mouse osteosarcoma cell lines, the consequences on the main cellular processes of RANK expression in osteosarcoma cells were analyzed. Results: The results revealed that RANK expression had no impact on cell proliferation and tumor growth, but stimulated cellular differentiation and, in an immune-compromised environment, increased the number of lung metastases. The analysis of RANKL, RANK and osteoprotegerin (OPG) expressions in biopsies of a cohort of patients revealed that while RANK expression in osteosarcoma cells was not significantly different between patients with or without metastases at the time of diagnosis, the OPG/RANK ratio decreased significantly. Conclusion: Altogether, these results are in favor of RANKL-RANK signaling inhibition as an adjuvant for the treatment of osteosarcoma.

. Validation of RANK forced-expression functionality in HOS cells by (A) qPCR analysis of RANK transcript expression, (B) Western blotting for RANK on 40 µg of total protein extract, (C) Western blotting for phosphorylated and total P38 on 40 µg of total protein extracted at different time-courses after addition of RANKL at 100 ng/mL and (D) flow cytometry analysis of RANK with AF682 antibody. The data showed that the Rank expression construction was well expressed (A) and induced significant expression of the encoded receptor at the cell surface (B and D). Moreover, this receptor was functional as attested by the rapid P38 activation following addition of RANKL to the cell culture medium (C). Figure S2. Injections of MOS-J A3N clone cells over-expressing RANK or not in C57BL/6 immune-competent and NMRI-nu immune-deficient mice. Tumor growth (A) and number of lung metastases (B) in NMRI nude mice after injection of three million A3N cells over-expressing RANK or not (7 mice in each group). A supplementary group (n = 4) injected with A3N cells over-expressing RANK was treated with an antibody blocking RANKL (IK22.5). The expression of RANK in A3N cells had no consequence on the tumor growth in the NMRI nude mice (A) but significantly increased the number of lung metastases (B). Moreover, this augmentation was reversed/prevented by the use of a RANKL blocking antibody (B). Tumor growth (C) and number of lung metastases (D) in C57BL/6 mice after injection of three million A3N cells over-expressing RANK or not (8 mice in each group). The expression of RANK in A3N cells had no consequence on either the tumor growth or the number of lung metastases in C57BL/6 mice. Growth curves (A and C) are shown as the mean ± SEM. All data analysis was performed using the Kruskal Wallis test: ns, not significant; * p < 0.05; ** p < 0.01. Figure S3. Analysis of the expression of RANK and Ki67 proteins using immunohistochemistry in sections of tumors induced by injections in NMRI-nu immune-deficient mice of HOS osteosarcoma cells that were native or over-expressing RANK. The RANK immunostaining appeared, as expected, more significant in the RANK overexpressing cells than in the parental cells while no difference in either the number of stained cells or the intensity of the staining was observed concerning the Ki67 immunostaining. Magnification ×200. Figure S4. Analysis by zymography of the impact of adding RANKL to the culture medium of native or RANK over-expressing HOS cells on the activity of metalloprotease 2 and 9. In the absence of RANKL addition, RANK over-expression in HOS cells induced an increase in MMP9 activity, while MMP2 activity was unchanged. The addition of RANKL had no impact on the activity of either enzymes in native HOS cells, where as in the RANK overexpressing HOS cells significant stimulation of the MMP9 activity was observed. MMP2activity did not appear to be modulated by adding RANKL to these cells. Figure S5. Impact of the host mice's genetic background on MOS-J PG1 tumor growth, the number of lung metastatic nodules and tumor osteoid tissue formation. MOS-JPG1 cells (3.106 cells) were injected into the vicinity of the tibias of 5-week-old Ranktg, Opg -/-, Rankl -/-and wild type mice in order to analyze the impact of different genetic backgrounds regarding elements in the RANKL/RANK/OPG triad onto the tumor growth (A), the number of lung nodules (B) and the tumor osteoid tissue formation (C). Tumor growth appeared to be slowed down only in the Rankl -/mice compared with the WT mice (A). Concerning the number of lung nodules, despite the longer time spent reaching the maximum ethically acceptable tumor volume (mouse sacrifice), Rank -/-mice revealed very few lung nodules compared to all other mice (B). For tumor osteoid tissue formation, significant apposition was observed in all genetic backgrounds in comparison with contralateral tibias (CT). However, this formation appeared less visible in osteolytic (Ranktg and Opg -/-) backgrounds compared to WT, and more visible in the osteopetrotic (Rankl -/-) background in relation to the different levels of osteoclast activity (C). n: number of mice. Tables   Table S1. Sequences of primers used for RT-qPCR analysis.

Lentivirus Production and Osteosarcoma Cell Transduction
Briefly, 6 × 10 6 HEK293FT cells (human embryonic kidney cells optimized for viral production) were seeded and transfected 24 h later with 3 µg of each packaging plasmid (ViraPower TM Packaging Mix, K497500; Invitrogen) and 9 µg of the transgene of interest (pEZ-Lv105-eGFP, EX-O0007-Lv105 and EX-Mm24198-Lv105); virus-containing supernatants were collected 48 h post-transfection and concentrated 60-fold by ultrafiltration. For titration, serial dilutions of the supernatants containing virus were tested on HEK293FT cells that were analyzed for EGFP expression 4 days post-infection using flow cytometry (FC500 flow cytometer, Beckman Coulter, Villepinte, France). The titers obtained were between 10 7 and 10 8 viral particles/µL. To generate stably modified HOS and MOS-J cell lines, 2 × 10 4 osteosarcoma cells were seeded in a 24-wells plate containing 300 µL of medium and infected with a multiplicity of infections of 50 particles/cell. After transduction, cells expressing the transgene were selected at confluence with 5 µg/mL puromycin (Gibco, Grand Island, NY, USA) to obtain a stable population. The eGFP and RANK expression levels were controlled using flow cytometry and RT-qPCR (for RANK).
PCR was performed on 1µL of gDNA from tails obtained with a Phire Animal Tissue Direct PCR Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Recombination of Rankl in T cells was checked after gDNA purification of splenic T-lymphocytes. Briefly, spleen was collected and maintained in cold isolation buffer (PBS with 2% FBS, 2mM EDTA) before being treated with a Dynabeads® FlowComp™ (Thermo Scientific) Mouse Pan T (CD90.2) kit according to the manufacturer's instructions. Before performing genomic DNA extraction using a PureLink® Genomic DNA Mini Kit, sample purity was verify using flow cytometry with anti CD3 immunostaining (Clone 17A2, ref555274, BD Biosciences, Franklin Lakes, NJ, USA). PCR Genotyping was then performed using Phire Hot Start II DNA Polymerase with the following primers: 5′-Lck: GTA ACA GTC GGC AGG AGT ATT TAT C and 3′-Lck: GGA CAG AGC CTC CCT GGT TAT GAA according to the manufacturer instructions.

Zymography Assay
After two days of culture in complete media, the cells were incubated for 24 h in serum-free media, in the presence or absence of sRANKL (100 ng/mL). After this culture period, MMP2 and MMP9 activities were analyzed in cell culture supernatants by zymography in 10 % SDS polyacrylamide gels containing 1 mg/mL gelatin (Sigma-Aldrich). Following electrophoresis, the gels were washed twice in 2.5% Triton X-100 for 30 min and incubated in 50 mM Tris-HCl, and 2 mM CaCl2 for 24 h at 37 °C. The following day, the gels were stained with 0.05% Coomassie brilliant blue (20% isopropanol, 10% acetic acid). Proteinase activity was visible as unstained regions. Gel pictures were taken using a G-box (Syngene, Cambridge, UK).

Flow Cytometry
The cells were harvested by incubation for 10min with a versene-EDTA solution (Lonza, Walkersville, MD, USA), followed by the addition of complete media to stop the reaction. 2 x 10 5 RANK-overexpressing cells or T-cells were stained with an anti-human RANK/TNFRSF11A