Delineating the metastatic process in osteosarcoma; the role of bone cells, the cell environment and autophagy

Osteosarcoma (OS) survival rate has plateaued over the past few decades, partly due to a paucity of research into mediators of the metastatic process. Recent evidence shows that targeting osteoclasts (OCs) with zoledronic acid (ZA) increases the number of metastatic lung lesions clinically, whereas treatment with Fulvestrant increases OC numbers and decreases metastatic lesions. This links OC function to OS metastasis. Through an in vitro model of OS metastasis, we have found that the highly metastatic OS cell line HOS-143B contains a subset of highly migratory spheroids with enhanced expression of stemness and metastasis-associated genes OCT3/4 (10-fold (p≤0.001)), NANOG (5-fold; p≤0.001), SOX2 (6-fold; p≤0.001), and CXCR4 (2-fold; p≤0.001) compared to monolayer cells. We have termed these spheroids with enhanced migratory capabilities ‘Migratory Bodies’ (MBs). We subsequently assessed the effect of OC-conditioned media on MB formation, where a significant reduction in MB formation in HOS-143B cells cultured with OC-conditioned media compared to control (43%; p≤0.001) or conditioned media from OCs treated with ZA (OC-ZA, 33%, p≤0.001) was observed. Furthermore, HOS-143B cells cultured with OC-conditioned media or OC-ZA-conditioned media exhibited divergent gene expression as assessed by RNA-sequencing. Differentially expressed genes were validated by RT-qPCR; MMP9 (2.4-fold decrease; p≤0.001), MMP14 (2-fold decrease; p≤0.01), CSF3 (2.5-fold decrease; p≤0.01) and SPOCK (1.6-fold decrease; p≤0.01). In an attempt to mimic OC-conditioned media, chemical inhibitors of MMP9 and STAT3 (downstream of CSF3) were tested in the MB metastasis assay. Both STAT3 and MMP9 expression is linked to lower survival in OS patients using the R2: Genomics Analysis and Visualization Platform. Inhibition of STAT3 and MMP9 significantly reduced MB formation from HOS-143B cells by 76% and 31% (p≤0.001) respectively, compared to control. Collectively, these data indicate that OC-conditioned media repress MB formation in highly metastatic HOS-143B cells. This supports our hypothesis that OS may reside in the bone environment due to interaction with bone cells i.e through secreted factors. It appears that disruption of this interaction leads to increased incidence of pulmonary metastasis. Lastly, our data suggests an approach to combat highly metastatic and migratory osteosarcoma cells by targeting STAT3 and/or MMP9.

Name dc:type.qualificationname

PhD

Level dc:type.qualificationlevel

PhD thesis

Grantor dc:publisher.institution

Middlesex University

Year dc:date.issued

2025

Author dc:creator

• Paternina Martinez, D.

Identifier

oai:repository.mdx.ac.uk:36862w

OAI identifier oai:identifier

oai:repository.mdx.ac.uk:36862w