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Exosomes as microenvironmental cue for engaging mesenchymal stem cells in osteosarcoma progression

Periodic Reporting for period 1 - S-OS (Exosomes as microenvironmental cue for engaging mesenchymal stem cells in osteosarcoma progression)

Reporting period: 2016-01-01 to 2017-12-31

Osteosarcoma (OS) is a very aggressive bone tumor that mainly affects children and adolescents. It is characterized by rapid growth and a strong tendency to recur and to form lung metastasis. In the presence of metastatic disease the prognosis is really poor and survival does not exceed 20%. The rarity, the heterogeneity and the genetic complexity of this type of tumor challenge the identification of therapeutic targets. As a consequence no improvements in OS treatment have been achieved in the last 30 year and novel therapeutic strategies are urgently needed.
Tumors are very dynamic structure in which tumor cells constantly communicate with surrounding normal cells and can even instruct them to support tumor growth and metastasis formation. Understanding and blocking these interactions might represent an effective strategy to stop cancer. Because OS occurs during the adolescent growth spurt and at sites of rapid bone growth, the local and systemic environment in which the tumor develops might have a crucial role in cancer development and progression.
In this project we explored the communication mediated by small extracellular vesicles (EVs) between tumor and normal stem cells residing in the bone marrow, called mesenchymal stem cells (MSC). We found that EVs secreted by highly malignant osteosarcoma cells alter the behavior of MSCs by inducing the production of proinflammatory factors. By releasing increased amounts of proinflammatory IL6, the educated MSCs promote OS growth and lung metastasis formation. Importantly, blockade of IL6 signaling with a therapeutic antibody abrogated the tumor-promoting effects of educated MSCs.
Our findings suggest that tumor EV are critical mediators of intercellular communication driving OS progression and provide the basis for testing immunomodulatory agents as new therapeutic options for osteosarcoma patients.
"My research activity has focused on the study of mesenchymal stem cell (MSC) function in cancer, and on how the interaction between malignant cells and MSCs promotes tumor progression. Through collaborations with multiple Departments at the hosting Institution and with international Institutions we developed a bioluminescent mouse model of osteosarcoma that enabled the study of the interactions between human OS cells and MSCs in vivo. The results of this investigation have been presented at international conferences and recently published in peer-reviewd journals (Baglio et al.,Clin Cancer Res 2017; Lagerweij et al., JoVE 2018; Bebelman et al., Pharmacology and Therapeutics 2018).

Exosomes are small extracellular vesicles (EVs) released by all cells that naturally function as communication devices by transferring proteins, lipids and regulatory RNAs between cells. We demonstrated that EVs released by OS cells trigger a prometastatic inflammatory loop by altering the behavior of MSCs. To investigate whether OS-released exosomes ""educate"" MSCs to support cancer progression, we developed a bioluminescent mouse model of osteosarcoma by inoculating osteosarcoma cells in the tibia of immunocompromised mice. We then educated human MSCs with exosomes released by OS cells, and injected tumor-educated (TEMSC) or non-educated MSCs into the tail vein of the osteosarcoma-bearing mice . We observed accelerated tumor growth in mice that received TEMSCs compared to the control groups. Moreover, we found MSCs in the tumor tissue and in the surrounding bone marrow, demonstrating that these cells can home to the tumor site . Importantly, administration of TEMSCs significantly increased the number of metastases compared to the control groups.

We showed that EVs released by the metastatic osteosarcoma cells instruct MSCs to produce increased levels of IL-6 and that this induction is dependent on a membrane-bound form of TGFβ specifically associated with the surface of the vesicles. We then asked whether we could inhibit tumor progression promoted by TEMSCs by using an IL-6 blocking agent. We found that administration of a clinically available anti-IL-6R antibody reduced tumor growth and metastasis formation in osteosarcoma-bearing mice receiving TEMSCs. Collectively, these data demonstrate that tumor EVs strongly influence MSCs behavior to promote cancer progression via the TGFβ-IL-6/STAT3 signaling axis and that we can interfere with this process using immunomodulatory drugs (Figure 1).

Finally, analysis of human osteosarcoma tissues revealed activation of IL-6/STAT3 signaling pathway and a strong TGFβ associated gene signature (Figure 4b) compared to normal bone tissue, confirming the clinical relevance of our pre-clinical findings. Moreover, we purified EVs from patient blood and showed that serum levels of EV-associated TGFβ are significantly higher in osteosarcoma patients compared to healthy control individuals. These data suggest that TGFβ-carrying vesicles of tumor origin act on stromal and tumor cells to sustain cancer progression, and could represent powerful biomarkers for disease monitoring.

The outcome of this project constitutes the basis of a new investigation that has recently received funding from the Dutch Cancer Society, in which we aim to further explore the communication between osteosarcoma and stromal immune cells and its clinical relevance. We are now setting up collaborations with pharmaceutical companies for testing specific combination of immunodulatory drugs, and with clinicians to evaluate the potential of EV-associated biomarkers to monitor cancer progression and treatment response.
Osteosarcoma is a highly malignant pediatric bone tumor. The main cause of death for the young osteosarcoma patients is the development of lung metastases. Undetectable micrometastases are estimated to be present in about 80% of patients at initial diagnosis, and only 20% survives for more than 5 years after initial diagnosis. The rarity, the high heterogeneity and the complex genomic rearrangements characterizing osteosarcoma precluded thus far the identification of molecular targets for therapeutic intervention. Consequently, despite the employment of different aggressive combination regimens, no major breakthrough has been achieved in the management of osteosarcoma in the last 30 years. Thus, there is a clear and urgent need for novel approaches to halt this devastating disease.
Treatment for osteosarcoma involves aggressive neoadjuvant multi-agent chemotherapy aimed at reducing the size of the tumor, followed by surgical resection of the primary tumor and post-operative chemotherapy. However, a consistent proportion of osteosarcoma patients with metastatic disease is resistant to this treatment, and no reliable methods to predict patient response currently exist. Thus, non-invasive tumor-associated biomarkers are urgently needed to improve prognosis and quality of life of osteosarcoma patients.
The outcome of our research activity lays the basis for novel therapeutic strategies based on the combination of antiflammatory drugs in osteosarcoma, and suggests that EVs may represent a valuable source of biomakers for disease monitoring and response to treatment.