The Osteosarcoma Microenvironment: A Complex But Targetable Ecosystem

Abstract

Osteosarcomas are the most frequent primary bone sarcomas, affecting mainly children, adolescents, and young adults, and with a second peak of incidence in elderly individuals. The current therapeutic management, a combined regimen of poly-chemotherapy and surgery, still remains largely insufficient, as patient survival has not improved in recent decades. Osteosarcomas are very heterogeneous tumors, both at the intra- and inter-tumor level, with no identified driver mutation. Consequently, efforts to improve treatments using targeted therapies have faced this lack of specific osteosarcoma targets. Nevertheless, these tumors are inextricably linked to their local microenvironment, composed of bone, stromal, vascular and immune cells and the osteosarcoma microenvironment is now considered to be essential and supportive for growth and dissemination. This review describes the different actors of the osteosarcoma microenvironment and gives an overview of the past, current, and future strategies of therapy targeting this complex ecosystem, with a focus on the role of extracellular vesicles and on the emergence of multi-kinase inhibitors.

Keywords: bone; extracellular vesicles; microenvironment; multi-kinase inhibitors; osteosarcoma; stromal cells; targeted therapies; vascular cells.

Figure 1

RANK–RANKL interactions under the canopy of bone remodeling compartments: old and new paradigms. The canopy is generated by bone lining cells and insulates a basic multicellular unit with osteoclasts (OC) and osteoblasts (OB). OC and OB precursors are recruited under the canopy, from respectively bone marrow- and blood stream-supplied hematopoietic stem cells and bone marrow-issued mesenchymal stromal cells. In the old and well-demonstrated paradigm, RANKL secreted by OB induces OC differentiation through RANK intracellular signaling (RANKL-RANK), while a new paradigm proposes a reverse signaling through RANKL intracellular signaling (RANK-RANKL) mediated by RANK-bearing extracellular vesicles EVs from OC [32]. RANK: receptor activator of nuclear factor kappa-B; RANKL: RANK ligand.

Figure 2

Proposed model of OPG/RANK–RANKL interactions in osteosarcoma (OS). RANK transduction induces the differentiation of osteoclasts (OC), leading to osteolysis, which in turn activates tumor cell proliferation, described as the vicious cycle. OPG is a decoy form of RANK, binding and neutralizing RANKL. Additionally, OPG increases proliferation of RANKL-expressing OS cells following its binding to an unknown receptor [38], possibly RANKL. The reverse signaling of RANKL, described recently in osteoblasts [32], could be also induced in OS cells. To the same extend, OS cells expressing RANKL could be activated by RANK-extracellular vesicles (EVs) produced by OC. RANK: receptor activator of nuclear factor kappa-B; RANKL: RANK ligand; OPG: osteoprotegerin.

Figure 3

Osteoblastic differentiation from mesenchymal stem/stromal cells (MSCs) to osteocytes. Transcription factor RUNX2 promotes MSC commitment toward the osteoblastic lineage at the early stages while repressing maturation in osteocytes. SP7 allows the differentiation of pre-osteoblasts into functional mature osteoblasts. RUNX2 induces expression of genes coding for expression of collagen type 1 (COL1), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCN) proteins. Transforming growth factor-β (TGF-β1) and WNT stimulate early stages of osteoblastic differentiation.

Figure 4

Histological analysis of experimentally induced osteosarcoma (OS) in athymic mice. Human OS-inducing cells (MNNG-HOS cells, CRL-1547, from American Type Culture Collection) were injected either alone (a,b) or co-injected with OS-derived stromal cells (OSDCs) (c,d) at tibial sites of athymic mice. Tumor samples were fixed in 10% buffered formaldehyde, embedded in paraffin wax, sectioned, and stained. Magnifications are indicated. (a) Human OS cells (brown nuclei) were distinguished from mouse cells (blue nuclei) by in situ hybridization using the human-specific repetitive Alu sequence [65]. Bone (B) spine lined by mouse cells was observed. (b) MNNG-HOS-induced tumor section was stained with hematoxylin–eosin–safran solution (HES). Tumor developed in muscle and appeared as an undifferentiated pleomorphic sarcoma (magnification in left panel), while osteoid matrix was observed only in intra-vascular tumor emboli (magnification in right panel). (c) Following co-injection with OSDC [64], MNNG-HOS-induced tumor was visible with abundant osteoid matrix surrounding anaplastic tumor cells. Tumor section was stained with HES. (d) Image of lung metastasis developed from co-injection of MNNG-HOS and OSDCs at paratibial site. Remarkable and non-usual immune cell infiltration (I) was observed in the vessel wall.

Figure 5

Sensing and modulating roles of mesenchymal stem/stromal cells (MSCs). MSCs constitutively express many mitogenic growth factors, chemokines, and matrix metalloproteinases at various levels. High/intermediate and low levels are represented by large or small font size, respectively. MSCs respond to tumor necrosis factor alpha (TNFα) by increasing expression (indicated in red) of some growth factor receptors (GFR), growth factors (GF), chemokine receptors (CR), chemokines, interleukins (IL), and matrix metalloproteinases (MMP) [61]. MSCs have autocrine and paracrine trophic properties, as all of these growth factors are able to act on several effectors of the tumor ecosystem (tumor, bone cells, endothelial cells, and immune cells). MSCs can also secrete extracellular vesicles (EVs) that convey diverse contents (see text for details), and they can receive information through EVs from bone pre-osteoclasts (pre-OC) and osteoclasts (OC) and tumor cells. EGFR: epithelial GFR; VEGFR: vascular endothelial GFR; TIE-1: angiopoietin receptor; TGFBR: transforming growth factor beta receptor; TIMP: tissue inhibitors of MMP; CXCL: chemokine (C-X-C motif) ligand; CCL: chemokine (C-C motif) ligand; GROα/β: growth-regulated oncogene alpha/beta (also known as CXCL1/2); MCP1/3: monocyte chemoattractant protein (also known as CCL2/7); SDF1: stromal cell-derived factor (also known as CXCL12); RANTES: Regulated on Activation Normal T Cell Expressed and Secreted (alias CCL5).