R38 - Investigating the role of osteosarcoma derived matrix-bound nanovesicles in promoting a cancerous phenotype in mesenchymal stem cells

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Catherine J. Zhu

Undergraduate Student
University of California, Santa Barbara
Goleta, United States

Co-Author(s)

AW

Andrew White

Graduate Student
UCSB, United States
Marley Dewey, PhD

Assistant Professor
University of California Santa Barbara
Santa Barbara, California, United States

Primary Investigator(s)

Marley Dewey, PhD

Assistant Professor
University of California Santa Barbara
Santa Barbara, California, United States

Introduction: : Osteosarcoma is a rare and malignant bone cancer with peak incidence in young adults. The average survival rate of osteosarcoma is approximately 59% but drops to 30% in metastatic cases. While new therapies (e.g. checkpoint inhibitors, CAR-T cells) have expanded treatment options for other cancers, the standard of care for osteosarcoma has remained largely unchanged for 40+ years, relying primarily on chemotherapy and surgical resection. Our project aims to understand the mechanisms driving osteosarcoma’s aggressive disease progression to advance outdated treatments and improve patient outcomes. One such mechanism involves extracellular vesicles (EVs)- lipid-bound nanoparticles secreted by all cells that mediate intercellular communication through bioactive cargo. EVs found in biofluids (liquid-EVs) are known to promote tumor growth and metastasis via crosstalk between malignant and healthy cells. Less explored are matrix-bound nanovesicles (MBVs)–a novel subtype of EV tethered to the extracellular matrix (ECM). Despite their integration in the ECM, the role of MBVs in cancer remains largely overlooked, even as ECM components like collagen and fibronectin are established contributors to cancer progression. To isolate MBV from osteosarcoma (SAOS-2) and mesenchymal stem cells, cells were cultured in 2D and 3D conditions (collagen biomaterial), and the properties of both groups of EVs were analyzed. Our study investigates the subsequent effects of 2D versus 3D culture-derived osteosarcoma MBVs on MSC cell viability, oncogene expression, and differentiation. By uncovering tumor-stroma communication, this work may identify EV components with potential as therapeutic targets or biomarkers for osteosarcoma.

Materials and

Methods: : EVs from particle-depleted conditioned media (liquid-EVs) and the ECM (MBVs) were isolated via size-exclusion chromatography (SEC) from SAOS-2 cells and MSCs cultured in either 2D polystyrene dishes or 3D type I collagen hydrogels over a period of 14 days. To release MBV from the ECM, it was digested with liberase. EV size, protein quantity, morphology, and surface markers (CD81, CD63) were analyzed by nanoparticle tracking analysis (NTA), microBCA, Transmission Electron Microscopy (TEM), and Western Blotting, respectively, before downstream applications. MSC (~6000 cells/cm2) were dosed every 3 days over a 12 day period with various concentrations (2 x 108, 2 x 107, 2 x 106, and 2 x 105 total particles) of SAOS-2 and MSC EVs (liquid, MBV). Cell metabolic activity was quantified by AlamarBlue assay . At the end of the experiment, Alizarin Red S staining was used to quantify mineral deposition, an osteoprotegerin ELISA to examine prevention of bone resorption, and RT-qPCR to measure expression levels of matrix metalloproteinase-9 (MMP9), tumor protein p53 (TP53), and MYC of cells in different treatment groups. An ordinary one-way ANOVA with Tukey post-hoc was used for statistical analysis between treatment groups and controls (media with PBS, media with DMSO).

Results, Conclusions, and Discussions::

Results:
All 2D EV samples had average particle sizes between 70-100 nm. EV morphology and general EV surface markers (CD81 for SAOS-2 EV, CD63 for MSC EV) were confirmed (Fig. 1). At day 3, there was no significant difference in metabolic activity between treatment groups and media control. However, at day 9, the MBV groups (SAOS-2 and MSC) both showed significantly greater cell numbers than liquid-EV groups when treated with the smallest EV dose (Fig. 2). Results for 3D EVs and endpoint assays (Alizarin Red, ELISA, RT-qPCR) are forthcoming.

Conclusions and Discussions:
Preliminary data confirms that EVs were properly isolated from both SAOS-2 and MSC cell lines and suggests that EV derived from 2D cultures may have a dose-dependent effect. Heightened cell proliferation occurred in groups treated with SAOS-2 MBV and MSC MBV from 2D cultures at the lowest dose (2 x 105) but did not occur in groups treated with the highest dose (2 x 108). This suggests that MBV may play a unique role in influencing cell behavior. Increased cellular proliferation/metabolic activity may be associated with uncontrolled cell growth, a hallmark of cancer. Further results will confirm these findings and demonstrate the impact of osteosarcoma-derived EV on MSC differentiation and oncogene expression. We are planning on conducting proteomic analyses of MSC and SAOS-2 derived EV, and future studies will also investigate the role of osteosarcoma-derived EV on cancer-associated fibroblasts to assess EV effects on metastasis. While there have been a lack of new treatments for osteosarcoma, new and more effective approaches can be developed with a complete understanding of the osteosarcoma microenvironment. Our findings could even have implications for the progression of other cancers beyond osteosarcoma. Insights into the role of MBVs on cancer biogenesis, as well as the effects of culture conditions on EV cargo, could inform the development of EV-based therapeutic tools for early cancer detection and targeted drug delivery.

Acknowledgements and/or References (Optional): : Thank you to Marley Dewey and Andrew White for their guidance on this project. Thank you to all other members of the Dewey Lab for their support. Thank you to CNSI and BioPacific for use of their facilities. This project was in part made possible by the Edu5 CIRM COMPASS program at UCSB.