Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis

Abstract

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10–20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

Extracellular vesicles secreted by highly metastatic clonal variants of osteosarcoma preferentially localize to the lungs and induce metastatic behaviour in poorly metastatic clones

“…This is consistent with published studies showing that commercial reagents or ultracentrifugation protocols produced EVs of equal purity and functional quality [14, 25–27]. Moreover, in a proteomic analysis of soluble factors secreted by metastatic and non-metastatic OS cells, we failed to detect VEGF, PDGF or TGFβ [28], thus excluding these as possible soluble contaminants in our EV preparation that could be contributing to the increase in metastasis. Thus, for the rest of our study we used a commercial precipitation kit (ExoQuick) for EV enrichment.…”

Microenvironment Signals and Mechanisms in the Regulation of Osteosarcoma

“…The levels of uPA and the uPA receptor (uPAR) were exclusively elevated in metastatic OS cells. These metastatic OS cells secrete both an active soluble form and an exosome-encapsulated form of uPA to drive the migration or metastatic conversion of OS cells [139]. Other research demonstrated that exosomes secreted by human MSCs could exhibit antiapoptotic function or cell-protective function to increase OS survival under serum starvation conditions [140].…”

The roles and implications of exosomes in sarcoma

“…Firstly, scientists distinguished a group of different osteosarcoma cell lines depending on their metastatic characteristics through in vivo and in vitro tests [65]. Among these metastatic osteosarcoma cell lines, KHOS cells were chosen as the exosomal donor cells because of their high expression of the uPA-uPAR axis (uPA and plasma membrane associated uPAR), which was proved to be exclusively associated with the metastatic behavior of osteosarcoma independent of Ras status [65]. Exosomes from KHOS cells were isolated through ultracentrifugation and confirmed by their particle diameter range (30-90 nm) and CD63 protein positive staining through dynamic light scattering and Western blot [65].…”

“…Among these metastatic osteosarcoma cell lines, KHOS cells were chosen as the exosomal donor cells because of their high expression of the uPA-uPAR axis (uPA and plasma membrane associated uPAR), which was proved to be exclusively associated with the metastatic behavior of osteosarcoma independent of Ras status [65]. Exosomes from KHOS cells were isolated through ultracentrifugation and confirmed by their particle diameter range (30-90 nm) and CD63 protein positive staining through dynamic light scattering and Western blot [65]. Meanwhile, uPA was detected both in exosomes and in the conditioned medium from tumors as the exosomal and soluble forms, respectively [65].…”

“…Exosomes from KHOS cells were isolated through ultracentrifugation and confirmed by their particle diameter range (30-90 nm) and CD63 protein positive staining through dynamic light scattering and Western blot [65]. Meanwhile, uPA was detected both in exosomes and in the conditioned medium from tumors as the exosomal and soluble forms, respectively [65]. Although further research about the relationship of the exosomes and metastasis in osteosarcoma has not been completed, it is still reasonable to conclude that exosomes are potential to influence metastatic behavior of osteosarcoma cells locally and at distant sites (Table 5).…”